Andrew J Bett

Publications of Andrew J Bett

• A trivalent recombinant Ad5 gag/pol/nef vaccine fails to protect rhesus macaques from infection or control virus replication after a limiting-dose heterologous SIV challenge.

  Authors: Matthew R Reynolds, Andrea M Weiler, Shari M Piaskowski, Michael Piatak, Henry T Robertson, David B Allison, Andrew J Bett, Danilo R Casimiro, John W Shiver, Nancy A Wilson, Jeffrey D Lifson, Wayne C Koff, David I Watkins

  Vaccine. 05/2012;

  It has been suggested that poor immunogenicity may explain the lack of vaccine efficacy in preventing or controlling HIV infection in the Step trial. To investigate this issue we vaccinated eightIt has been suggested that poor immunogenicity may explain the lack of vaccine efficacy in preventing or controlling HIV infection in the Step trial. To investigate this issue we vaccinated eight Indian rhesus macaques with a trivalent replication-incompetent adenovirus serotype 5 vaccine expressing SIV Gag, Pol, and Nef using a regimen similar to that employed in the Step trial. We detected broad vaccine-induced CD8(+) (2-7 pool-specific responses) and CD4(+) (5-19 pool-specific responses) T-cell responses in IFN-γ ELISPOT assays at one week post-boost using fresh PBMC. However, using cryopreserved cells at one and four weeks post-boost we observed a reduction in both the number and magnitude of most vaccine-induced responses. This demonstrates that the time points and conditions chosen to perform immune assays may influence the observed breadth and frequency of vaccine-induced T-cell responses. To evaluate protective efficacy, we challenged the immunized macaques, along with naïve controls, with repeated, limiting doses of the heterologous swarm isolate SIVsmE660. Vaccination did not significantly affect acquisition or control of virus replication in vaccinees compared to naïve controls. Post-infection we observed an average of only two anamnestic CD8(+) T-cell responses per animal, which may not have been sufficiently broad to control heterologous virus replication. While the trivalent vaccine regimen induced relatively broad T-cell responses in rhesus macaques, it failed to protect against infection or control viral replication. Our results are consistent with those observed in the Step trial and indicate that SIV immunization and challenge studies in macaque models of HIV infection can be informative in assessing pre-clinical HIV vaccines.

• An optimized SIV DNA vaccine can serve as a boost for Ad5 and provide partial protection from a high-dose SIVmac251 challenge.

  Authors: Natalie A Hutnick, Devin J F Myles, Lauren Hirao, Veronica L Scott, Bernadette Ferraro, Amir S Khan, Mark G Lewis, Christopher J Miller, Andrew J Bett, Danilo Casimiro, Niranjan Y Sardesai, J Joseph Kim, John Shiver, David B Weiner

  Vaccine. 03/2012;

  One limitation in the development of an improved cellular response needed for an effective HIV-vaccine is the inability to induce robust effector T-cells capable of suppressing a heterologousOne limitation in the development of an improved cellular response needed for an effective HIV-vaccine is the inability to induce robust effector T-cells capable of suppressing a heterologous challenge. To improve cellular immune responses, we examined the ability of an optimized DNA vaccine to boost the cellular immune responses induced by a highly immunogenic Ad5 prime. Five Chinese rhesus macaques received pVax encoding consensus (con) gag/pol/env intramuscularly (IM) with electroporation followed by the Merck Ad5 gag/pol/nef vaccine. A second group of five animals were vaccinated with Merck Ad5 gag/pol/nef followed by pVax gag/pol/env. One year following vaccination, Ad5-prime DNA-boosted monkeys and four unvaccinated controls received an intrarectal challenge with 1000 ID50 SIV(mac)251. The quality and magnitude of the T-cell response was analyzed by ELISpot and polyfunctional flow cytometry. We observed that an Ad5-prime DNA-boost resulted in significantly elevated SIV-specific T-cell responses even compared with animals receiving a DNA-prime Ad5-boost. Ad5 prime DNA boosted animals were capable of suppressing a pathogenic SIV(mac)251 challenge. Peak control correlated with the expansion of HLA-DR(+) CD8(+) T-cells two weeks post-infection. These data illustrate that high optimization of a DNA vaccine can drive of immune responses primed by a robust vector system. This previously unachievable feature of these newly optimized DNAs warrants future studies of this strategy that may circumvent issues of serology associated with viral vector prime-boost systems.

• Low-dose penile SIVmac251 exposure of rhesus macaques infected with adenovirus type 5 (Ad5) and then immunized with a replication-defective Ad5-based SIV gag/pol/nef vaccine recapitulates the results of the phase IIb step trial of a similar HIV-1 vaccine.

  Authors: Huma Qureshi, Zhong-Min Ma, Ying Huang, Gregory Hodge, Michael A Thomas, Janet DiPasquale, Veronique DeSilva, Linda Fritts, Andrew J Bett, Danilo R Casimiro, John W Shiver, Marjorie Robert-Guroff, Michael N Robertson, Michael B McChesney, Peter B Gilbert, Christopher J Miller

  Journal of virology. 12/2011; 86(4):2239-50.

  The Step Trial showed that the MRKAd5 HIV-1 subtype B Gag/Pol/Nef vaccine did not protect men from HIV infection or reduce setpoint plasma viral RNA (vRNA) levels but, unexpectedly, it did modestlyThe Step Trial showed that the MRKAd5 HIV-1 subtype B Gag/Pol/Nef vaccine did not protect men from HIV infection or reduce setpoint plasma viral RNA (vRNA) levels but, unexpectedly, it did modestly enhance susceptibility to HIV infection in adenovirus type 5 (Ad5)-seropositive, uncircumcised men. As part of the process to understand the results of the Step Trial, we designed a study to determine whether rhesus macaques chronically infected with a host-range mutant Ad5 (Ad5hr) and then immunized with a replication defective Ad5 SIVmac239 Gag/Pol/Nef vaccine were more resistant or susceptible to SIV infection than unimmunized rhesus macaques challenged with a series of escalating dose penile exposures to SIVmac 251. The Ad5 SIV vaccine induced CD8(+) T cell responses in 70% of the monkeys, which is similar to the proportion of humans that responded to the vaccine in the Step Trial. However, the vaccine did not protect vaccinated animals from penile SIV challenge. At the lowest SIV exposure dose (10(3) 50% tissue culture infective doses), 2 of 9 Ad5-seropositive animals immunized with the Ad5 SIV vaccine became infected compared to 0 of 34 animals infected in the other animal groups (naive animals, Ad5-seropositive animals immunized with the empty Ad5 vector, Ad5-seronegative animals immunized with the Ad5 SIV vaccine, and Ad5-seronegative animals immunized with the empty Ad5 vector). Penile exposure to more concentrated virus inocula produced similar rates of infection in all animal groups. Although setpoint viral loads were unaffected in Step vaccinees, the Ad5 SIV-immunized animals had significantly lower acute-phase plasma vRNA levels compared to unimmunized animals. Thus, the results of the nonhuman primate (NHP) study described here recapitulate the lack of protection against HIV acquisition seen in the Step Trial and suggest a greater risk of infection in the Ad5-seropositive animals immunized with the Ad5 SIV vaccine. Further studies are necessary to confirm the enhancement of virus acquisition and to discern associated mechanisms.

• Transcriptional profiling of vaccine-induced immune responses in humans and non-human primates.

  Authors: I-Ming Wang, Andrew J Bett, Razvan Cristescu, Andrey Loboda, Jan Ter Meulen

  Microbial biotechnology. 11/2011; 5(2):177-87.

  There is an urgent need for pre-clinical and clinical biomarkers predictive of vaccine immunogenicity, efficacy and safety to reduce the risks and costs associated with vaccine development. ResultsThere is an urgent need for pre-clinical and clinical biomarkers predictive of vaccine immunogenicity, efficacy and safety to reduce the risks and costs associated with vaccine development. Results emerging from immunoprofiling studies in non-human primates and humans demonstrate clearly that (i) type and duration of immune memory are largely determined by the magnitude and complexity of the innate immune signals and (ii) genetic signatures highly predictive of B-cell and T-cell responses can be identified for specific vaccines. For vaccines with similar composition, e.g. live attenuated viral vaccines, these signatures share common patterns. Signatures predictive of vaccine efficacy have been identified in a few experimental challenge studies. This review aims to give an overview of the current literature on immunoprofiling studies in humans and also presents some of our own data on profiling of licensed and experimental vaccines in non-human primates.

• The development of recombinant subunit envelope-based vaccines to protect against dengue virus induced disease.

  Authors: Beth-Ann G Coller, David E Clements, Andrew J Bett, Sangeetha L Sagar, Jan H Ter Meulen

  Vaccine. 07/2011; 29(42):7267-75.

  Challenges associated with the interference observed between the dengue virus components within early tetravalent live-attenuated vaccines led many groups to explore the development of recombinantChallenges associated with the interference observed between the dengue virus components within early tetravalent live-attenuated vaccines led many groups to explore the development of recombinant subunit based vaccines. Initial efforts in the field were hampered by low yields and/or improper folding, but the use of the Drosophila S2 cell expression system provided a mechanism to overcome these limitations. The truncated dengue envelope proteins (DEN-80E) for all four dengue virus types are expressed in the S2 system at high levels and have been shown to maintain native-like conformation. The DEN-80E proteins are potent immunogens when formulated with a variety of adjuvants, inducing high titer virus neutralizing antibody responses and demonstrating protection in both mouse and non-human primate models. Tetravalent vaccine formulations have shown no evidence of immune interference between the four DEN-80E antigens in preclinical models. Based on the promising preclinical data, the recombinant DEN-80E proteins have now advanced into clinical studies. An overview of the relevant preclinical data for these recombinant proteins is presented in this review.

• Synthesis and immunological activities of novel Toll-like receptor 7 and 8 agonists.

  Authors: Ekambar R Kandimalla, Mary Struthers, Andrew J Bett, Thomas Wisniewski, Sheri A Dubey, Weiwen Jiang, Melissa Precopio, Zhenhua Sun, Hao Wang, Tao Lan, Sudhir Agrawal, Danilo R Casimiro

  Cellular immunology. 04/2011; 270(2):126-34.

  Single-stranded oligoribonucleotides (ORNs) stimulate innate immune responses through TLR7 and TLR8. Specific linkages and chemical modifications incorporated into synthetic ORN can greatly enhanceSingle-stranded oligoribonucleotides (ORNs) stimulate innate immune responses through TLR7 and TLR8. Specific linkages and chemical modifications incorporated into synthetic ORN can greatly enhance nuclease stability, selectivity, and potency. In the present study, we have synthesized 15 ORN containing different sequence compositions and chemical modifications and studied their TLR7- and TLR8-mediated immune response profiles in HEK293 cells expressing human TLR7 or TLR8, human PBMCs, mDCs and pDCs, non-human primate (NHP) PBMCs, and in vivo in mice and NHPs. Based on the results obtained, eight of the ORNs containing specific chemical modifications induced immune responses through both TLR7 and TLR8, including activation of NF-κB in TLR7- and TLR8-transfected cell lines; induction of IFN-α, IL-6, TNF-α, IL-12, and IP-10 in human PBMCs; IFN-α induction in human pDCs; CD80 upregulation in human pDCs and mDCs; IL-12 induction following acute administration in mice; IFN-α, IP-10, IL-6, and IL-12 induction in NHP PBMCs; and IFN-α, IP-10, and IL-6 induction following acute administration in NHPs. Seven of the ORNs show selectivity for TLR8-induced responses; they specifically activate only TLR8-transfected cell lines, induce cytokines other than IFN-α in human and NHP PBMCs, activate mDCs more than pDCs, and do not induce IL-12 acutely in mice, consistent with the lack of functional TLR8 in mice. The novel TLR8-selective ORNs also induce cytokines other than IFN-α acutely in NHPs. In conclusion, we have designed and synthesized novel ORNs with varying sequence compositions and chemical modifications, which selectively act as agonists of TLR8 or dual agonists of TLR7 and TLR8.

• Comparison of T cell immune responses induced by vectored HIV vaccines in non-human primates and humans.

  Authors: Andrew J Bett, Sheri A Dubey, Devan V Mehrotra, Liming Guan, Romnie Long, Kiersten Anderson, Kelly Collins, Christine Gaunt, Rose Fernandez, Suzanne Cole, Steve Meschino, Aimin Tang, Xiao Sun, Sanjay Gurunathan, Jim Tartaglia, Michael N Robertson, John W Shiver, Danilo R Casimiro

  Vaccine. 10/2010; 28(50):7881-9.

  Following the disappointing outcome of the phase IIb test-of-concept step study in which Merck's adenovirus type 5 (Ad5) HIV-1 clade B gag/pol/nef vaccine failed to demonstrate efficacy in HIVFollowing the disappointing outcome of the phase IIb test-of-concept step study in which Merck's adenovirus type 5 (Ad5) HIV-1 clade B gag/pol/nef vaccine failed to demonstrate efficacy in HIV high-risk individuals, an extensive review of the trial and preclinical studies which supported the trial is ongoing. One point of interest is how well preclinical nonhuman primate immunogenicity studies predicted what was observed in humans. Here we compare the HIV-1-specific cellular immune responses elicited in nonhuman primates and human clinical trial subjects to several HIV-1 vaccine candidates. We find that although rhesus macaques are immunologically more responsive to vaccination than humans, the hierarchy in potency of single-modality prime-boost regimens using several vector approaches (adenovirus, DNA, and pox vectors) was well predicted. Vaccine approaches using complex formulations such as novel adjuvants (DNA+CRL1005) or mixed-modality prime-boost (DNA/Ad5; Ad5/ALVAC) did not correlate as well between rhesus macaques and humans. Although the immunogenicity of the vaccines and vaccine regimens evaluated were not all accurately predicted, testing in rhesus macaques generally offers an indispensable tool for ranking the immunological potential of HIV-1 vaccine candidates.

• Comparative analysis of immune responses induced by vaccination with SIV antigens by recombinant Ad5 vector or plasmid DNA in rhesus macaques.

  Authors: Lauren A Hirao, Ling Wu, Abhishek Satishchandran, Amir S Khan, Ruxandra Draghia-Akli, Adam C Finnefrock, Andrew J Bett, Michael R Betts, Danilo R Casimiro, Niranjan Y Sardesai, J Joseph Kim, John W Shiver, David B Weiner

  Molecular therapy : the journal of the American Society of Gene Therapy. 08/2010; 18(8):1568-76.

  DNA vaccines have undergone important enhancements in their design, formulation, and delivery process. Past literature supports that DNA vaccines are not as immunogenic in nonhuman primates as liveDNA vaccines have undergone important enhancements in their design, formulation, and delivery process. Past literature supports that DNA vaccines are not as immunogenic in nonhuman primates as live vector systems. The most potent recombinant vector system for induction of cellular immune responses in macaques and humans is adenovirus serotype 5 (Ad5), an important benchmark for new vaccine development. Here, we performed a head-to-head evaluation of the Merck Ad5 SIV vaccine and an optimized electroporation (EP) delivered SIV DNA vaccine in macaques. Animals receiving the Ad5 vaccine were immunized three times, whereas the DNA-vaccinated animals were immunized up to four times based on optimized protocols. We observed significant differences in the quantity of IFNgamma responses by enzyme-linked immunosorbent spot (ELISpot), greater proliferative capacity of CD8(+) T cells, and increased polyfunctionality of both CD4(+) and CD8(+) T cells in the DNA-vaccinated group. Importantly, Ad5 immunizations failed to boost following the first immunization, whereas DNA responses were continually boosted with all four immunizations demonstrating a major advantage of these improved DNA vaccines. These optimized DNA vaccines induce very different immune phenotypes than traditional Ad5 vaccines, suggesting that they could play an important role in vaccine research and development.

• Synthesis and immunological activities of novel agonists of toll-like receptor 9.

  Authors: Mary Struthers, Andrew J Bett, Thomas Wisniewski, Sheri A Dubey, Melissa Precopio, Weiwen Jiang, Zhenhua Sun, Hao Wang, Ireneusz Nowak, Mallikarjuna R Putta, Dong Yu, Jimmy X Tang, Ekambar R Kandimalla, Sudhir Agrawal, Danilo R Casimiro

  Cellular immunology. 03/2010; 263(1):105-13.

  Novel agonists of TLR9 with two 5'-ends and synthetic immune stimulatory motifs, referred to as immune modulatory oligonucleotides (IMOs) are potent agonists of TLR9. In the present study, we haveNovel agonists of TLR9 with two 5'-ends and synthetic immune stimulatory motifs, referred to as immune modulatory oligonucleotides (IMOs) are potent agonists of TLR9. In the present study, we have designed and synthesized 15 novel IMOs by incorporating specific chemical modifications and studied their immune response profiles both in vitro and in vivo. Analysis of the immunostimulatory profiles of these IMOs in human and NHP cell-based assays suggest that changes in the number of synthetic immunostimulatory motifs gave only a subtle change in immune stimulation of pDCs as indicated by IFN-alpha production and pDC maturation while the addition of self-complementary sequences produced more dramatic changes in both pDC and B cell stimulation. All IMOs induced cytokine production in vivo immediately after administration in mice. Representative compounds were also compared for the ability to stimulate cytokine production in vivo (IFN-alpha and IP-10) in rhesus macaques after intra-muscular administration.

• Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors.

  Authors: Miguel Aste-Amézaga, Ningyan Zhang, Janet E Lineberger, Beth A Arnold, Timothy J Toner, Mingcheng Gu, Lingyi Huang, Salvatore Vitelli, Kim T Vo, Peter Haytko [......] Kenneth N Ross, Laura L Franlin, Hui Wang, Fubao Wang, Michael Chastain, Andrew J Bett, Laurent P Audoly, Jon C Aster, Stephen C Blacklow, Hans E Huber

  PloS one. 01/2010; 5(2):e9094.

  Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signalingNotch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target. Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50) values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors. Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.

• Robust, Vaccine-Induced CD8+ T Lymphocyte Response against an Out-of-Frame Epitope.

  Authors: Nicholas J Maness, Nancy A Wilson, Jason S Reed, Shari M Piaskowski, Jonah B Sacha, Andrew D Walsh, Elizabeth Thoryk, Gwendolyn J Heidecker, Michael P Citron, Xiaoping Liang, Andrew J Bett, Danilo R Casimiro, David I Watkins

  Journal of immunology (Baltimore, Md. : 1950). 11/2009;

  Rational vaccines designed to engender T cell responses require intimate knowledge of how epitopes are generated and presented. Recently, we vaccinated 8 Mamu-A *02(+) rhesus macaques with every SIVRational vaccines designed to engender T cell responses require intimate knowledge of how epitopes are generated and presented. Recently, we vaccinated 8 Mamu-A *02(+) rhesus macaques with every SIV protein except Envelope (Env). Surprisingly, one of the strongest T cell responses engendered was against the Env protein, the Mamu-A *02-restricted epitope, Env(788-795)RY8. In this paper, we show that translation from an alternate reading frame of both the Rev-encoding DNA plasmid and the rAd5 vector engendered Env(788-795)RY8-specific CD8(+) T cells of greater magnitude than "normal" SIV infection. Our data demonstrate both that the pathway from vaccination to immune response is not well understood and that products of alternate reading frames may be rich and untapped sources of T cell epitopes.

• Safety and Immunogenicity of the MRKAd5 and MRKAd6 HIV-1 Trigene Vaccines Alone and in Combination in Healthy Adults.

  Authors: Clayton Harro, Xiao Sun, Jon E Stek, Randi Y Leavitt, Devan V Mehrotra, Fubao Wang, Andrew J Bett, Danilo R Casimiro, John W Shiver, Mark J DiNubile, Erin Quirk

  Clinical and vaccine immunology : CVI. 07/2009;

  Background: Preexisting immunity to adenovirus (Ad) type 5 diminishes immune responses to vaccines using Ad5 as a vector. Alternate Ad serotypes as vaccine vectors might overcome Ad5-specificBackground: Preexisting immunity to adenovirus (Ad) type 5 diminishes immune responses to vaccines using Ad5 as a vector. Alternate Ad serotypes as vaccine vectors might overcome Ad5-specific neutralizing antibodies and enhance immune responses in populations with a high prevalence of Ad5 immunity. Methods: Healthy HIV-seronegative adults were enrolled in a blinded, randomized, dose-escalating, placebo-controlled study. In Part A, subjects with baseline Ad6 titers </=18 received the MRKAd6 HIV-1 trigene vaccine at Weeks 0, 4, and 26. In Part B, subjects stratified by Ad5 titers (</=200 or >200) and Ad6 titers (</=18 or >18) received MRKAd5+6 HIV-1 trigene vaccine at Weeks 0, 4, and 26. Immunogenicity was assessed by ELISPOT at Week 30. Results: No serious adverse events occurred. MRKAd6 trigene vaccine recipients responded more often to Nef than Gag or Pol. In Part A, ELISPOT response rates to >/=2 vaccine antigens were 14%, 63%, and 71% at the 10(9), 10(10), and 10(11) vg/dose, respectively. All responders had a positive Nef-specific ELISPOT. In Part B, Nef-ELISPOT response rates at 10(10) vg/dose of the MRKAd5+6 trigene vaccine were 50% in low Ad5/low Ad6 stratum (n=8), 78% in low Ad5/high Ad6 stratum (n=9), 75% in the high Ad5/low Ad6 stratum (n=8), and 44% in the high Ad5/high Ad6 stratum (n=9). Conclusions: The MRKAd6 and MRKAd5+6 trigene vaccines elicited dose-dependent responses predominantly to Nef and were generally well tolerated, indicating that Ad6 should be considered a candidate vector for future vaccines. Although small sample sizes limit conclusions drawn from this exploratory study, combining two Ad vectors may be a useful vaccine strategy to circumvent isolated immunity to a single Ad type.

• Vaccine-induced Cellular Responses Control SIV Replication After Heterologous Challenge.

  Authors: Nancy A Wilson, Brandon F Keele, Jason S Reed, Shari M Piaskowski, Caitlin E Mac Nair, Andrew J Bett, Xiaoping Liang, Fubao Wang, Elizabeth Thoryk, Gwendolyn J Heidecker [......] Eva G Rakasz, Stephen Erickson, David B Allison, Michael Piatak, Jeffrey D Lifson, John W Shiver, Danilo R Casimiro, George M Shaw, Beatrice H Hahn, David I Watkins

  Journal of virology. 04/2009;

  All HIV vaccine efficacy trials to date have ended in failure. Structural features of the Env glycoprotein and its enormous variability have frustrated efforts to induce broadly-reactive neutralizingAll HIV vaccine efficacy trials to date have ended in failure. Structural features of the Env glycoprotein and its enormous variability have frustrated efforts to induce broadly-reactive neutralizing antibodies. To explore the extent to which vaccine-induced cellular immune responses, in the absence of neutralizing antibodies, can control replication of a heterologous, mucosal viral challenge, we vaccinated eight macaques with a DNA/Ad5 regimen expressing all of the proteins of SIVmac239 except Env. Vaccinees mounted high-frequency T cell responses against 11-34 epitopes. We challenged the vaccinees and eight naïve animals with the heterologous biological isolate SIVsmE660, using a regimen intended to mimic typical human HIV exposures resulting in infection. Viral loads in the vaccinees were significantly less at both peak (1.9 log reduction p<0.03) and at set point (3.3 log reduction p<0.003) than those of control naïve animals. Five of eight vaccinated macaques controlled acute peak viral replication to less than 80,000 vRNA copy Eq/ml and to less than 100 vRNA copy Eq/ml in the chronic phase. Our results demonstrate that broad vaccine-induced cellular immune responses can effectively control replication of a pathogenic, heterologous AIDS virus, suggesting that T cell based vaccines may have greater potential than previously appreciated.

• Attenuation of simian immunodeficiency virus SIVmac239 infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing Gag.

  Authors: Danilo R Casimiro, Fubao Wang, William A Schleif, Xiaoping Liang, Zhi-Qiang Zhang, Timothy W Tobery, Mary-Ellen Davies, Adrian B McDermott, David H O'Connor, Arthur Fridman [......] Anthony Carella, Kara S Punt, Kara J Sykes, Lingyi Huang, Virginia I Ausensi, Margaret Bachinsky, Usha Sadasivan-Nair, David I Watkins, Emilio A Emini, John W Shiver

  Journal of virology. 01/2006; 79(24):15547-55.

  The prophylactic efficacy of DNA and replication-incompetent adenovirus serotype 5 (Ad5) vaccine vectors expressing simian immunodeficiency virus (SIV) Gag was examined in rhesus macaques using anThe prophylactic efficacy of DNA and replication-incompetent adenovirus serotype 5 (Ad5) vaccine vectors expressing simian immunodeficiency virus (SIV) Gag was examined in rhesus macaques using an SIVmac239 challenge. Cohorts of either Mamu-A*01(+) or Mamu-A*01(-) macaques were immunized with a DNA prime-Ad5 boost regimen; for comparison, a third cohort consisting of Mamu-A*01(+) monkeys was immunized using the Ad5 vector alone for both prime and boost. All animals, along with unvaccinated control cohorts of Mamu-A*01(+) and Mamu-A*01(-) macaques, were challenged intrarectally with SIVmac239. Viral loads were measured in both peripheral and lymphoid compartments. Only the DNA prime-Ad5-boosted Mamu-A*01(+) cohort exhibited a notable reduction in peak plasma viral load (sevenfold) as well as in early set-point viral burdens in both plasma and lymphoid tissues (10-fold) relative to those observed in the control monkeys sharing the same Mamu-A*01 allele. The degree of control in each animal correlated with the levels of Gag-specific immunity before virus challenge. However, virus control was short-lived, and indications of viral escape were evident as early as 6 months postinfection. The implications of these results in vaccine design and clinical testing are discussed.

• Serotype specificity of adenovirus purification using anion-exchange chromatography.

  Authors: John O Konz, Rebecca C Livingood, Andrew J Bett, Aaron R Goerke, Michael E Laska, Sangeetha L Sagar

  Human gene therapy. 12/2005; 16(11):1346-53.

  Recombinant adenoviruses continue to be a leading vector choice for gene transfer applications, with growing interest in the use of less prevalent serotypes, and of chimeras. As a result, theRecombinant adenoviruses continue to be a leading vector choice for gene transfer applications, with growing interest in the use of less prevalent serotypes, and of chimeras. As a result, the development of scaleable purification processes for alternative serotypes is needed. Anion-exchange chromatography is routinely used for scaleable adenovirus type 5 purification; however, retention varies for other serotypes because of differences in the exposed capsid proteins. Understanding how the viral surface influences retention behavior can provide a rational basis for chromatography development and optimization. In this work, chimeric vectors were used to show that the hexon protein is responsible for retention differences in anion-exchange chromatography. Next, the relative retention of eight serotypes from three subgroups was studied. Although all serotypes bound to the anion-exchange resin, the sodium chloride required to elute the virus varied over a 2- fold range, from 270 to 490 mM. Retention was accurately correlated to the electrostatic properties of the hexon protein, with an average error in sodium chloride concentration required to elute of only 14 mM. This correlation enables preparative chromatography gradients for alternative serotypes to be established with minimal effort.

• Heterologous human immunodeficiency virus type 1 priming-boosting immunization strategies involving replication-defective adenovirus and poxvirus vaccine vectors.

  Authors: Danilo R Casimiro, Andrew J Bett, Tong-Ming Fu, Mary-Ellen Davies, Aimin Tang, Keith A Wilson, Minchun Chen, Romnie Long, Troy McKelvey, Michael Chastain, Sanjay Gurunathan, Jim Tartaglia, Emilio A Emini, John Shiver

  Journal of virology. 11/2004; 78(20):11434-8.

  We compared the human immunodeficiency virus type 1 (HIV-1)-specific cellular immune responses elicited in nonhuman primates by HIV-1 gag-expressing replication-defective adenovirus serotype 5 (Ad5)We compared the human immunodeficiency virus type 1 (HIV-1)-specific cellular immune responses elicited in nonhuman primates by HIV-1 gag-expressing replication-defective adenovirus serotype 5 (Ad5) or poxvirus vectors, used either alone or in combination with each other. The responses arising from a heterologous Ad5 priming-poxvirus boosting regimen were significantly greater than those elicited by homologous regimens with the individual vectors or by a heterologous poxvirus priming-Ad5 boosting regimen. The heterologous Ad5 priming-poxvirus boosting approach may have potential utility in humans as a means of inducing high levels of cellular immunity.

• Quantitative adenovirus neutralization assays based on the secreted alkaline phosphatase reporter gene: application in epidemiologic studies and in the design of adenovector vaccines.

  Authors: Miguel Aste-Amézaga, Andrew J Bett, Fubao Wang, Danilo R Casimiro, Joseph M Antonello, Deepa K Patel, Elayne C Dell, Laura L. Franlin, Nancy M Dougherty, Philip S Bennett, Helen C Perry, Mary-Ellen Davies, John W Shiver, Paul M Keller, Mark D Yeager

  Human gene therapy. 04/2004; 15(3):293-304.

  Replication-defective recombinant adenoviruses (rAd) are used as vectors for vaccines as well as for gene therapy. To determine type-specific antibodies to adenovirus (Ad) serotypes 2, 5, 24, 34, andReplication-defective recombinant adenoviruses (rAd) are used as vectors for vaccines as well as for gene therapy. To determine type-specific antibodies to adenovirus (Ad) serotypes 2, 5, 24, 34, and 35, we developed quantitative neutralization assays using recombinant adenoviruses with the secreted alkaline phosphatase (SEAP) reporter gene. Among the standardized parameters, the concentration of infectious and noninfectious adenoviral particles used in the assay is critical for a reliable comparison of data from different studies. The usefulness of this assay was demonstrated in a pilot epidemiologic study of 40 healthy individuals. In this study, the highest prevalence of antiadenovirus antibodies was found for the Ad2 serotype (82.5%), followed by Ad5 (35%). The prevalence of antiadenovirus antibodies for the serotypes 24, 34, and 35 was low (7.5%, 2.5%, and 0%, respectively). In addition, epidemiologic parameters such as gender and age were statistically evaluated. A positive association was found between age and the presence of anti-Ad5 antibodies. The assay was also useful for evaluating the presence of antiadenovirus antibodies in the design of vaccines using a rhesus monkey model. In this animal model, it was possible to determine differential dose and time responses, and the specificity for the detection of neutralizing antibodies was assessed. The evaluation of serotype-specific neutralizing antibodies can be of both clinical and epidemiologic importance as a means of selecting the appropriate serotype adenovector(s).

• Comparative analysis of the effects of packaging signal, transgene orientation, promoters, polyadenylation signals, and E3 region on growth properties of first-generation adenoviruses.

  Authors: Rima Youil, Timothy J Toner, Qin Su, Danilo Casimiro, John W Shiver, Ling Chen, Andrew J Bett, Bethany M Rogers, Eric C Burden, Aimin Tang, Michelle Chen, Emilio A Emini, David C Kaslow, John G Aunins, Nedim E Altaras

  Human gene therapy. 08/2003; 14(10):1017-34.

  First-generation adenovectors have been developed for gene therapy and vaccine applications. The construction of these adenovectors has entailed the use of numerous types of expression cassettes. ItFirst-generation adenovectors have been developed for gene therapy and vaccine applications. The construction of these adenovectors has entailed the use of numerous types of expression cassettes. It has long been known that first-generation adenovectors can be rescued more easily and to higher titers with some transgenes than with others. This study has systematically shown that there can be marked differences in growth properties of recombinant adenovectors attributable to the use of promoters, the orientation of the transgene within the E1A/E1B-deleted region, and the inclusion of the E3 region. In addition, we had demonstrated the benefit of extending the packaging signal region to include elements V, VI, and VII. The effects of the complete packaging region were studied by plasmid competition studies between original and modified adenovectors. Similar competition studies between E3(+) and E3(-) adenovectors were performed and showed that the E3(+) vector had a growth advantage over its E3(-) counterpart. By making various changes, we have enhanced the growth capacity of our recombinant adenovector by more than 3-fold under serum-free and cell suspension growth conditions. Along with this enhanced growth, our adenovectors have maintained their genetic stability after 21 successive passages in cell culture. This increased robustness will be critical when adapting first-generation recombinant adenovectors to commercial production.

• Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene.

  Authors: Danilo R Casimiro, Ling Chen, Tong-Ming Fu, Robert K Evans, Michael J Caulfield, Mary-Ellen Davies, Aimin Tang, Minchun Chen, Lingyi Huang, Virginia Harris [......] Natasha Persaud, Timothy J Toner, Qin Su, Xiaoping Liang, Rima Youil, Michael Chastain, Andrew J Bett, David B Volkin, Emilio A Emini, John W Shiver

  Journal of virology. 07/2003; 77(11):6305-13.

  Cellular immune responses, particularly those associated with CD3(+) CD8(+) cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection withCellular immune responses, particularly those associated with CD3(+) CD8(+) cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defective adenovirus serotype 5 (Ad5) vector, each expressing the same codon-optimized HIV-1 gag gene for immunogenicity in rhesus monkeys. The DNA vaccines were formulated with and without one of two chemical adjuvants (aluminum phosphate and CRL1005). The Ad5-gag vector was the most effective in eliciting anti-Gag CTL. The vaccine produced both CD4(+) and CD8(+) T-cell responses, with the latter consistently being the dominant component. To determine the effect of existing antiadenovirus immunity on Ad5-gag-induced immune responses, monkeys were exposed to adenovirus subtype 5 that did not encode antigen prior to immunization with Ad5-gag. The resulting anti-Gag T-cell responses were attenuated but not abolished. Regimens that involved priming with different DNA vaccine formulations followed by boosting with the adenovirus vector were also compared. Of the formulations tested, the DNA-CRL1005 vaccine primed T-cell responses most effectively and provided the best overall immune responses after boosting with Ad5-gag. These results are suggestive of an immunization strategy for humans that are centered on use of the adenovirus vector and in which existing adenovirus immunity may be overcome by combined immunization with adjuvanted DNA and adenovirus vector boosting.

• Hexon gene switch strategy for the generation of chimeric recombinant adenovirus.

  Authors: Rima Youil, Timothy J Toner, Qin Su, Minchun Chen, Aimin Tang, Andrew J Bett, Danilo Casimiro

  Human gene therapy. 02/2002; 13(2):311-20.

  The usefulness of adenovirus as a vehicle for transgene delivery is limited greatly by the induction of neutralizing anti-adenoviral immunity following the initial administration, thereby resultingThe usefulness of adenovirus as a vehicle for transgene delivery is limited greatly by the induction of neutralizing anti-adenoviral immunity following the initial administration, thereby resulting in shorter-term and reduced levels of transgene expression. In this paper, we outline a strategy for the generation of recombinant Ad5-based adenovectors that have undergone a complete hexon exchange in an effort to circumvent pre-existing anti-vector humoral immunity. Eighteen different chimeric adenoviral vectors (from subgroups A, B, C, D, and E) have been constructed using a combination of direct cloning and bacterial homologous recombination methods. However, only chimeric Ad5-based constructs in which the hexons from Ad1, Ad2, Ad6, and Ad12 are incorporated in place of the Ad5 hexon were successfully rescued into viruses. Despite several attempts, the remaining fourteen chimeric adenovectors were not rescuable. In vivo rodent studies using transgenes for human immunodeficiency virus type 1 (HIV-1) gag and secreted human alkaline phosphatase (SEAP) suggest that the Ad5/Ad6-gag chimera (wherein Ad5 hexon was replaced with that of Ad6) is able to evade neutralizing antibodies generated against Ad5 vector efficiently. However, it appears that cross-reactive cytotoxic T lymphocytes (CTL) may also play a role in controlling in vivo infectivity of Ad5/Ad6-gag chimera. The Ad5/Ad12 chimera was found to be extremely ineffective in the i.m. delivery and expression of HIV-1 gag in mice compared to the Ad5/Ad6 construct. Implications of these results will be discussed.