John W Shiver

Publications of John W Shiver

• 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.

• 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.

• Clostridium difficile and methicillin-resistant Staphylococcus aureus: emerging concepts in vaccine development.

  Authors: David C Kaslow, John W Shiver

  Annual review of medicine. 01/2011; 62:201-15.

  Both Clostridium difficile and Staphylococcus aureus asymptomatically colonize a significant percentage of humans, particularly during the first year of life. The epidemiology of both has been andBoth Clostridium difficile and Staphylococcus aureus asymptomatically colonize a significant percentage of humans, particularly during the first year of life. The epidemiology of both has been and continues to be quite dynamic; presently, we are in the midst of epidemics of infections by C. difficile and S. aureus. These ancient microbes are now armed with more potent virulence factors, which have extended their reach from the hospital into community settings, and from the elderly and debilitated hosts into the younger and otherwise healthy population. This review presents some emerging concepts that will likely shape efforts to develop active and passive immunization interventions in response to the reemergence of these bacterial pathogens.

• Mapping HIV-1 vaccine induced T-cell responses: bias towards less-conserved regions and potential impact on vaccine efficacy in the Step study.

  Authors: Fusheng Li, Adam C Finnefrock, Sheri A Dubey, Bette T M Korber, James Szinger, Suzanne Cole, M Juliana McElrath, John W Shiver, Danilo R Casimiro, Lawrence Corey, Steven G Self

  PloS one. 01/2011; 6(6):e20479.

  T cell directed HIV vaccines are based upon the induction of CD8+ T cell memory responses that would be effective in inhibiting infection and subsequent replication of an infecting HIV-1 strain, aT cell directed HIV vaccines are based upon the induction of CD8+ T cell memory responses that would be effective in inhibiting infection and subsequent replication of an infecting HIV-1 strain, a process that requires a match or near-match between the epitope induced by vaccination and the infecting viral strain. We compared the frequency and specificity of the CTL epitope responses elicited by the replication-defective Ad5 gag/pol/nef vaccine used in the Step trial with the likelihood of encountering those epitopes among recently sequenced Clade B isolates of HIV-1. Among vaccinees with detectable 15-mer peptide pool ELISpot responses, there was a median of four (one Gag, one Nef and two Pol) CD8 epitopes per vaccinee detected by 9-mer peptide ELISpot assay. Importantly, frequency analysis of the mapped epitopes indicated that there was a significant skewing of the T cell response; variable epitopes were detected more frequently than would be expected from an unbiased sampling of the vaccine sequences. Correspondingly, the most highly conserved epitopes in Gag, Pol, and Nef (defined by presence in >80% of sequences currently in the Los Alamos database www.hiv.lanl.gov) were detected at a lower frequency than unbiased sampling, similar to the frequency reported for responses to natural infection, suggesting potential epitope masking of these responses. This may be a generic mechanism used by the virus in both contexts to escape effective T cell immune surveillance. The disappointing results of the Step trial raise the bar for future HIV vaccine candidates. This report highlights the bias towards less-conserved epitopes present in the same vaccine used in the Step trial. Development of vaccine strategies that can elicit a greater breadth of responses, and towards conserved regions of the genome in particular, are critical requirements for effective T-cell based vaccines against HIV-1. Trial registration: ClinicalTrials.gov NCT00849680, A Study of Safety, Tolerability, and Immunogenicity of the MRKAd5 Gag/Pol/Nef Vaccine in Healthy Adults.

• 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.

• Role of nonhuman primates in the evaluation of candidate AIDS vaccines: an industry perspective.

  Authors: Silvija I Staprans, Mark B Feinberg, John W Shiver, Danilo R Casimiro

  Current opinion in HIV and AIDS. 09/2010; 5(5):377-85.

  To consider how nonhuman primate (NHP) model systems can best contribute to HIV vaccine development. We review the traditional roles of NHP model systems in vaccine development and compare this withTo consider how nonhuman primate (NHP) model systems can best contribute to HIV vaccine development. We review the traditional roles of NHP model systems in vaccine development and compare this with how NHP models have been used in HIV vaccine research and development. Comparisons of the immune responses elicited by cellular immune response-inducing vaccines in macaques and humans illustrate the value of primate studies for the relative ranking of HIV vaccine concepts for their likely immunogenicity in humans. The unusual structures (e.g. long complementarity-determining regions) of known broadly neutralizing HIV antibodies (bNAbs) suggest that it is critical to test candidate env immunogens in NHPs, whose germline antibody repertoires resemble those of humans. Recent clinical efficacy trial results question the utility of existing NHP challenge models in predicting HIV vaccine efficacy in humans, and highlight the need to further develop models in which acquisition of infection can be reliably evaluated. When evaluated in models using low virus dose challenges that better approximate human sexual exposure to HIV - some vaccine and passive NAb interventions appear to protect against acquisition of infection. NHP models have important roles in the preclinical evaluation, optimization, and ranking of novel HIV immunogens. The apparent vaccine efficacy observed using low virus dose challenge models provides an opportunity to investigate the correlates of protection.

• Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge.

  Authors: Gayathri Bommakanti, Michael P Citron, Robert W Hepler, Cheryl Callahan, Gwendolyn J Heidecker, Tariq Ahmad Najar, Xianghan Lu, Joseph G Joyce, John W Shiver, Danilo R Casimiro, Jan ter Meulen, Xiaoping Liang, Raghavan Varadarajan

  Proceedings of the National Academy of Sciences of the United States of America. 08/2010; 107(31):13701-6.

  Influenza HA is the primary target of neutralizing antibodies during infection, and its sequence undergoes genetic drift and shift in response to immune pressure. The receptor binding HA1 subunit ofInfluenza HA is the primary target of neutralizing antibodies during infection, and its sequence undergoes genetic drift and shift in response to immune pressure. The receptor binding HA1 subunit of HA shows much higher sequence variability relative to the metastable, fusion-active HA2 subunit, presumably because neutralizing antibodies are primarily targeted against the former in natural infection. We have designed an HA2-based immunogen using a protein minimization approach that incorporates designed mutations to destabilize the low pH conformation of HA2. The resulting construct (HA6) was expressed in Escherichia coli and refolded from inclusion bodies. Biophysical studies and mutational analysis of the protein indicate that it is folded into the desired neutral pH conformation competent to bind the broadly neutralizing HA2 directed monoclonal 12D1, not the low pH conformation observed in previous studies. HA6 was highly immunogenic in mice and the mice were protected against lethal challenge by the homologous A/HK/68 mouse-adapted virus. An HA6-like construct from another H3 strain (A/Phil/2/82) also protected mice against A/HK/68 challenge. Regions included in HA6 are highly conserved within a subtype and are fairly well conserved within a clade. Targeting the highly conserved HA2 subunit with a bacterially produced immunogen is a vaccine strategy that may aid in pandemic preparedness.

• 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.

• Vaccination with peptide mimetics of the gp41 prehairpin fusion intermediate yields neutralizing antisera against HIV-1 isolates.

  Authors: Elisabetta Bianchi, Joseph G Joyce, Michael D Miller, Adam C Finnefrock, Xiaoping Liang, Marco Finotto, Paolo Ingallinella, Philip McKenna, Michael Citron, Elizabeth Ottinger, Robert W Hepler, Renee Hrin, Deborah Nahas, Chengwei Wu, David Montefiori, John W Shiver, Antonello Pessi, Peter S Kim

  Proceedings of the National Academy of Sciences of the United States of America. 06/2010; 107(23):10655-60.

  Eliciting a broadly neutralizing polyclonal antibody response against HIV-1 remains a major challenge. One approach to vaccine development is prevention of HIV-1 entry into cells by blocking theEliciting a broadly neutralizing polyclonal antibody response against HIV-1 remains a major challenge. One approach to vaccine development is prevention of HIV-1 entry into cells by blocking the fusion of viral and cell membranes. More specifically, our goal is to elicit neutralizing antibodies that target a transient viral entry intermediate (the prehairpin intermediate) formed by the HIV-1 gp41 protein. Because this intermediate is transient, a stable mimetic is required to elicit an immune response. Previously, a series of engineered peptides was used to select a mAb (denoted D5) that binds to the surface of the gp41 prehairpin intermediate, as demonstrated by x-ray crystallographic studies. D5 inhibits the replication of HIV-1 clinical isolates, providing proof-of-principle for this vaccine approach. Here, we describe a series of peptide mimetics of the gp41 prehairpin intermediate designed to permit a systematic analysis of the immune response generated in animals. To improve the chances of detecting weak neutralizing polyclonal responses, two strategies were employed in the initial screening: use of a neutralization-hypersensitive virus and concentration of the IgG fraction from immunized animal sera. This allowed incremental improvements through iterative cycles of design, which led to vaccine candidates capable of generating a polyclonal antibody response, detectable in unfractionated sera, that neutralize tier 1 HIV-1 and simian HIV primary isolates in vitro. Our findings serve as a starting point for the design of more potent immunogens to elicit a broadly neutralizing response against the gp41 prehairpin intermediate.

• Efficacy of multivalent adenovirus-based vaccine against simian immunodeficiency virus challenge.

  Authors: Danilo R Casimiro, Kara Cox, Aimin Tang, Kara J Sykes, Meizhen Feng, Fubao Wang, Andrew Bett, William A Schleif, Xiaoping Liang, Jessica Flynn [......] Jing Lin, Deepa Patel, Mary-Ellen Davies, Gwendolyn J Heidecker, Daniel C Freed, Sheri Dubey, David H O'Connor, David I Watkins, Zhi-Qiang Zhang, John W Shiver

  Journal of virology. 03/2010; 84(6):2996-3003.

  The prophylactic efficacies of several multivalent replication-incompetent adenovirus serotype 5 (Ad5) vaccines were examined in rhesus macaques using an intrarectal high-dose simian immunodeficiencyThe prophylactic efficacies of several multivalent replication-incompetent adenovirus serotype 5 (Ad5) vaccines were examined in rhesus macaques using an intrarectal high-dose simian immunodeficiency virus SIVmac239 challenge model. Cohorts of Mamu-A*01(+)/B*17(-) Indian rhesus macaques were immunized with one of several combinations of Ad5 vectors expressing Gag, Pol, Nef, and Env gp140; for comparison, a Mamu-A*01(+) cohort was immunized using the Ad5 vector alone. There was no sign of immunological interference between antigens in the immunized animals. In general, expansion of the antigen breadth resulted in more favorable virological outcomes. In particular, the order of efficacy trended as follows: Gag/Pol/Nef/Env approximately Gag/Pol > Gag approximately Gag/Pol/Nef > Nef. However, the precision in ranking the vaccines based on the study results may be limited by the cohort size, and as such, may warrant additional testing. The implications of these results in light of the recent discouraging results of the phase IIb study of the trivalent Ad5 HIV-1 vaccine are discussed.

• International epidemiology of human pre-existing adenovirus (Ad) type-5, type-6, type-26 and type-36 neutralizing antibodies: Correlates of high Ad5 titers and implications for potential HIV vaccine trials.

  Authors: T Christopher Mast, Lisa Kierstead, Swati B Gupta, Alexander A Nikas, Esper G Kallas, Vladimir Novitsky, Bernard Mbewe, Punee Pitisuttithum, Mauro Schechter, Eftyhia Vardas, Nathan D Wolfe, Miguel Aste-Amezaga, Danilo R Casimiro, Paul Coplan, Walter L Straus, John W Shiver

  Vaccine. 11/2009;

  Replication-defective adenoviruses have been utilized as candidate HIV vaccine vectors. Few studies have described the international epidemiology of pre-existing immunity to adenoviruses. We enrolledReplication-defective adenoviruses have been utilized as candidate HIV vaccine vectors. Few studies have described the international epidemiology of pre-existing immunity to adenoviruses. We enrolled 1904 participants in a cross-sectional serological survey at seven sites in Africa, Brazil, and Thailand to assess neutralizing antibodies (NA) for adenovirus types Ad5, Ad6, Ad26 and Ad36. Clinical trial samples were used to assess NA titers from the US and Europe. The proportions of participants that were negative were 14.8% (Ad5), 31.5% (Ad6); 41.2% (Ad26) and 53.6% (Ad36). Adenovirus NA titers varied by geographic location and were higher in non-US and non-European settings, especially Thailand. In multivariate logistic regression analysis, geographic setting (non-US and non-European settings) was statistically significantly associated with having higher Ad5 titers; participants from Thailand had the highest odds of having high Ad5 titers (adjusted OR=3.53, 95% CI: 2.24, 5.57). Regardless of location, titers of Ad5NA were the highest and Ad36 NA were the lowest. Coincident Ad5/6 titers were lower than either Ad5 or Ad6 titers alone. Understanding pre-existing immunity to candidate vaccine vectors may contribute to the evaluation of vaccines in international populations.

• Comparative Cell-Mediated Immunogenicity of DNA/DNA, DNA/Adenovirus Type 5 (Ad5), or Ad5/Ad5 HIV-1 Clade B gag Vaccine Prime-Boost Regimens.

  Authors: David M Asmuth, Elizabeth L Brown, Mark J DiNubile, Xiao Sun, Carlos Del Rio, Clayton Harro, Michael C Keefer, James G Kublin, Sheri A Dubey, Lisa S Kierstead, Danilo R Casimiro, John W Shiver, Michael N Robertson, Erin K Quirk, Devan V Mehrotra

  The Journal of infectious diseases. 11/2009;

  Background. We report composite results from the Merck phase I program of near-consensus clade B human immunodeficiency virus (HIV) type 1 gag vaccines. Methods. Healthy HIV-uninfected adults wereBackground. We report composite results from the Merck phase I program of near-consensus clade B human immunodeficiency virus (HIV) type 1 gag vaccines. Methods. Healthy HIV-uninfected adults were enrolled in 6 blinded placebo-controlled studies evaluating the immunogenicity of (1) a 4-dose regimen of a DNA vaccine, (2) a 3-dose priming regimen of the DNA vaccine with a booster dose of an adenovirus type 5 (Ad5)-vectored vaccine, or (3) a 3-dose regimen of the Ad5 vaccine. The DNA plasmid was provided with or without an aluminum phosphate or CRL1005 adjuvant. The primary end point was the unfractionated HIV-1 gag-specific interferon gamma enzyme-linked immunospot (ELISpot) response 4 weeks after the final dose. Results. Overall, 254 (83%) of 307 subjects randomized to the vaccine groups were evaluable. Adjuvants did not enhance immunogenicity of the DNA vaccine. Postboost ELISpot responder frequencies were higher for Ad5-containing regimens than for the DNA/DNA regimen (33%) but were similar for DNA/Ad5 (55%) and Ad5/Ad5 (50%). DNA/DNA elicited mainly a CD4 response, whereas Ad5/Ad5 elicited mainly a CD8 response; DNA/Ad5 generated CD4 and CD8 responses comparable to those of DNA/DNA and Ad5/Ad5, respectively. Conclusions. The DNA vaccine alone or as a priming regimen for the Ad5 vaccine did not increase unfractionated ELISpot responses compared with the Ad5 vaccine alone. Qualitative T cell responses to different vaccine regimens deserve further study.

• Adenovirus-specific immunity after immunization with an Ad5 HIV-1 vaccine candidate in humans.

  Authors: Kara L O'Brien, Jinyan Liu, Sharon L King, Ying-Hua Sun, Joern E Schmitz, Michelle A Lifton, Natalie A Hutnick, Michael R Betts, Sheri A Dubey, Jaap Goudsmit, John W Shiver, Michael N Robertson, Danilo R Casimiro, Dan H Barouch

  Nature medicine. 09/2009; 15(8):873-5.

  The immunologic basis for the potential enhanced HIV-1 acquisition in adenovirus serotype 5 (Ad5)-seropositive individuals who received the Merck recombinant Ad5 HIV-1 vaccine in the STEP studyThe immunologic basis for the potential enhanced HIV-1 acquisition in adenovirus serotype 5 (Ad5)-seropositive individuals who received the Merck recombinant Ad5 HIV-1 vaccine in the STEP study remains unclear. Here we show that baseline Ad5-specific neutralizing antibodies are not correlated with Ad5-specific T lymphocyte responses and that Ad5-seropositive subjects do not develop higher vector-specific cellular immune responses as compared with Ad5-seronegative subjects after vaccination. These findings challenge the hypothesis that activated Ad5-specific T lymphocytes were the cause of the potential enhanced HIV-1 susceptibility in the STEP study.

• 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.

• Comparative immunogenicity evaluations of influenza A virus M2 peptide as recombinant virus like particle or conjugate vaccines in mice and monkeys.

  Authors: Tong-Ming Fu, Karen M Grimm, Michael P Citron, Daniel C Freed, Jiang Fan, Paul M Keller, John W Shiver, Xiaoping Liang, Joseph G Joyce

  Vaccine. 02/2009;

  Immunization against M2 peptide, also called M2e, from influenza A virus is an innovative vaccine approach for induction of cross-strain protective immunity. Two promising M2 vaccine compositionsImmunization against M2 peptide, also called M2e, from influenza A virus is an innovative vaccine approach for induction of cross-strain protective immunity. Two promising M2 vaccine compositions reported to date are M2 peptide chemically conjugated to carrier proteins or M2 peptide recombinantly expressed on the surface of virus like particles (VLPs) of hepatitis B virus core antigen (HBVc). To conduct a head-to-head comparison of these approaches, we constructed two recombinant HBVc VLPs expressing M2 peptide and prepared two conjugate vaccines with M2 peptide chemically coupled to Neisseria meningitidis outer membrane complex (OMPC) or HBVc VLP, respectively. Here, we showed superior immunogenicity of M2 peptide conjugated to OMPC and M2 peptide expressed on the surface of HBVc antigen based on dose-titration responses in mice. Surprisingly, HBVc expressing M2 peptide was an inferior vaccine in rhesus monkeys, whether as a primary vaccine or as a booster vaccine, when compared with M2-OMPC conjugate vaccine.

• Synthetic peptide vaccines: the quest to develop peptide vaccines for influenza, HIV and Alzheimer's disease.

  Authors: Elisabetta Bianchi, Paolo Ingallinella, Marco Finotto, Joseph Joyce, Xiaoping Liang, Michael D Miller, Gene G Kinney, Gennaro Ciliberto, John W Shiver, Antonello Pessi

  Advances in experimental medicine and biology. 02/2009; 611:121-3.

• Characterizations of four monoclonal antibodies against M2 protein ectodomain of influenza A virus.

  Authors: Tong-Ming Fu, Daniel C Freed, Melanie S Horton, Jiang Fan, Michael P Citron, Joseph G Joyce, Victor M Garsky, Danilo R Casimiro, Qinjian Zhao, John W Shiver, Xiaoping Liang

  Virology. 01/2009;

  M2 protein of influenza A virus has been implicated as a target for vaccines with broad cross-strain coverage. Studies in small animal models have shown that antibody responses induced by 23-mer M2M2 protein of influenza A virus has been implicated as a target for vaccines with broad cross-strain coverage. Studies in small animal models have shown that antibody responses induced by 23-mer M2 peptide vaccines can provide protection against influenza A virus challenge. To study antiviral mechanisms of Merck M2-OMPC conjugate vaccine, we generated and characterized four M2 peptide-specific monoclonal antibodies (mAbs). Here we demonstrated that the protection by our M2 mAbs is independent of NK-mediated effector functions in mice. The protective mAbs preferentially bind to M2 multimers composed of two or more M2 peptides in parallel orientation. Our findings indicate that the protective M2 Ab prefer to bind to epitopes located within the N-terminal 10 amino acids of the M2 peptide, and the epitopes are likely formed by two M2 peptides in parallel orientation. The implications of these results in antiviral mechanisms of immune responses induced by M2 vaccines are discussed.

• PD-1 blockade in rhesus macaques: impact on chronic infection and prophylactic vaccination.

  Authors: Adam C Finnefrock, Aimin Tang, Fengsheng Li, Daniel C Freed, Meizhen Feng, Kara S Cox, Kara J Sykes, James P Guare, Michael D Miller, David B Olsen, Daria J Hazuda, John W Shiver, Danilo R Casimiro, Tong-Ming Fu

  Journal of immunology (Baltimore, Md. : 1950). 01/2009; 182(2):980-7.

  Programmed Cell Death 1 (PD-1) plays a crucial role in immunomodulation. Binding of PD-1 to its ligand receptors down-regulates immune responses, and published reports suggest that this immuneProgrammed Cell Death 1 (PD-1) plays a crucial role in immunomodulation. Binding of PD-1 to its ligand receptors down-regulates immune responses, and published reports suggest that this immune modulation is exploited in cases of tumor progression or chronic viral infection to evade immune surveillance. Thus, blockade of this signal could restore or enhance host immune functions. To test this hypothesis, we generated a panel of mAbs specific to human PD-1 that block PD ligand 1 and tested them for in vitro binding, blocking, and functional T cell responses, and evaluated a lead candidate in two in vivo rhesus macaque (Macaca mulatta) models. In the first therapeutic model, chronically SIV-infected macaques were treated with a single infusion of anti-PD-1 mAb; viral loads increased transiently before returning to, or falling below, pretreatment baselines. In the second prophylactic model, naive macaques were immunized with an SIV-gag adenovirus vector vaccine. Induced PD-1 blockade caused a statistically significant (p<0.05) increase in the peak percentage of T cells specific for the CM9 Gag epitope. These new results on PD-1 blockade in nonhuman primates point to a broader role for PD-1 immunomodulation and to potential applications in humans.

• Safety and Immunogenicity of Adenovirus-Vectored Near-Consensus HIV Type 1 Clade B gag Vaccines in Healthy Adults.

  Authors: Clayton D Harro, Michael N Robertson, Michelle A Lally, Lori D O'Neill, Srilatha Edupuganti, Paul A Goepfert, Mark J Mulligan, Frances H Priddy, Sheri A Dubey, Lisa S Kierstead, Xiao Sun, Danilo R Casimiro, Mark J DiNubile, John W Shiver, Randi Y Leavitt, Devan V Mehrotra

  AIDS research and human retroviruses. 12/2008;

  Abstract Vaccines inducing pathogen-specific cell-mediated immunity are being developed using attenuated adenoviral (Ad) vectors. We report the results of two independent Phase I trials of similarAbstract Vaccines inducing pathogen-specific cell-mediated immunity are being developed using attenuated adenoviral (Ad) vectors. We report the results of two independent Phase I trials of similar replication-deficient Ad5 vaccines containing a near-consensus HIV-1 clade B gag transgene. Healthy HIV-uninfected adults were enrolled in two separate, multicenter, dose-escalating, blinded, placebo-controlled studies to assess the safety and immunogenicity of a three-dose homologous regimen of Ad5 and MRKAd5 HIV-1 gag vaccines given on day 1, week 4, and week 26. Adverse events were collected for 29 days following each intradeltoid injection. The primary immunogenicity endpoint was the proportion of subjects with a positive unfractionated Gag-specific IFN-gamma ELISPOT response measured 4 weeks after the last dose (week 30). Analyses were performed after combining data for each dose group from both protocols, stratifying by baseline Ad5 titers. Overall, 252 subjects were randomized to receive either vaccine or placebo, including 229 subjects (91%) who completed the study through week 30. Tolerability and immunogenicity did not appear to differ between the Ad5 and MRKAd5 vaccines. The frequency of injection-site reactions was dose dependent. Systemic adverse events were also dose dependent and more frequent in subjects with baseline Ad5 titers <200 versus >/=200, especially after the first dose. The percent of ELISPOT responders and the ELISPOT geometric means overall were significantly higher for all four vaccine doses studied compared to placebo, and were generally higher in vaccine recipients with baseline Ad5 titers <200 versus >/=200. Ad5 titers increased after vaccination in a dose-dependent fashion. Both Ad5-vectored HIV-1 vaccines were generally well tolerated and induced cell-mediated immune responses against HIV Gag-peptides in the majority of healthy adults with baseline Ad5 titers <200. Preexistent and/or vaccine-induced immunity to the Ad5 vector may dampen the CMI response to HIV Gag.