Danilo R Casimiro

Publications of Danilo R Casimiro

• 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.

• Human adenovirus-specific T cells modulate HIV-specific T cell responses to an Ad5-vectored HIV-1 vaccine.

  Authors: Nicole Frahm, Allan C DeCamp, David P Friedrich, Donald K Carter, Olivier D Defawe, James G Kublin, Danilo R Casimiro, Ann Duerr, Michael N Robertson, Susan P Buchbinder, Yunda Huang, Gregory A Spies, Stephen C De Rosa, M Juliana McElrath

  The Journal of clinical investigation. 12/2011; 122(1):359-67.

  Recombinant viruses hold promise as vectors for vaccines to prevent infectious diseases with significant global health impacts. One of their major limitations is that preexisting anti-vectorRecombinant viruses hold promise as vectors for vaccines to prevent infectious diseases with significant global health impacts. One of their major limitations is that preexisting anti-vector neutralizing antibodies can reduce T cell responses to the insert antigens; however, the impact of vector-specific cellular immunity on subsequent insert-specific T cell responses has not been assessed in humans. Here, we have identified and compared adenovirus-specific and HIV-specific T cell responses in subjects participating in two HIV-1 vaccine trials using a vaccine vectored by adenovirus serotype 5 (Ad5). Higher frequencies of pre-immunization adenovirus-specific CD4⁺ T cells were associated with substantially decreased magnitude of HIV-specific CD4⁺ T cell responses and decreased breadth of HIV-specific CD8⁺ T cell responses in vaccine recipients, independent of type-specific preexisting Ad5-specific neutralizing antibody titers. Further, epitopes recognized by adenovirus-specific T cells were commonly conserved across many adenovirus serotypes, suggesting that cross-reactivity of preexisting adenovirus-specific T cells can extend to adenovirus vectors derived from rare serotypes. These findings provide what we believe to be a new understanding of how preexisting viral immunity may impact the efficacy of vaccines under current evaluation for prevention of HIV, tuberculosis, and malaria.

• 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.

• Evaluating functional antibodies in rhesus monkeys immunized with hepatitis B virus surface antigen vaccine with novel adjuvant formulations.

  Authors: Daniel C Freed, Victoria M Towne, Danilo R Casimiro, Qinjian Zhao, Tong-Ming Fu

  Vaccine. 11/2011; 29(50):9385-90.

  Effective and safe novel adjuvants are of great interest to the vaccine research community. In this study, we describe our evaluation of adjuvant formulations containing a TLR9 agonist adjuvantEffective and safe novel adjuvants are of great interest to the vaccine research community. In this study, we describe our evaluation of adjuvant formulations containing a TLR9 agonist adjuvant (ISS1018) or ISCOMATRIX™ adjuvant for a two-dose regimen of hepatitis B virus surface antigen virus-like particle vaccine in mice and rhesus macaques. Our results show a 10-20 fold improvement in Ab binding titers determined in an antigen-sandwich assay for adjuvant formulations with ISCOMATRIX™ adjuvant, in comparison to routine aluminum formulation. Furthermore, we optimized a competition assay to evaluate a functional component of immune sera, using a conformation-dependent and protective mAb, RFHBs1, as the probe. Although good correlation was observed between Ab binding titers from the antigen-sandwich assay and functional titers from the in-solution competition against RFHBs1, the latter assessment provided a much more stringent ranking of adjuvant formulations than the former. These results indicate the importance of evaluating functional Abs when assessing and comparing novel adjuvant formulations, as it provides another angle to investigate the effects of change in adjuvant composition on antigenic integrity of the testing vaccines.

• Quantitative analysis of neutralizing antibody response to human cytomegalovirus in natural infection.

  Authors: Dai Wang, Fengsheng Li, Daniel C Freed, Adam C Finnefrock, Aimin Tang, Shannon N Grimes, Danilo R Casimiro, Tong-Ming Fu

  Vaccine. 09/2011; 29(48):9075-80.

  Naturally acquired immunity significantly reduces the risk of congenital cytomegalovirus (CMV) infection in future pregnancies. An immune response comparable to that of natural infection has beenNaturally acquired immunity significantly reduces the risk of congenital cytomegalovirus (CMV) infection in future pregnancies. An immune response comparable to that of natural infection has been used as a benchmark for CMV vaccine efficacy; however, the magnitude and persistence of the neutralizing antibody responses in naturally infected women are not completely understood. In this study, we quantitatively analyzed a panel of 375 female CMV convalescent sera ranging in age from 18 to 84 years, for its ability to block virus entry into epithelial cells and fibroblasts, as well as its binding potential to CMV particles. The geometric mean titer of the sera in this panel to neutralize 50% of the virus entry into epithelial cells was 7491, compared to 802 for entry into fibroblasts. The epithelial neutralizing titers were statistically indistinguishable among different age groups, and conformed to a normal distribution. There was a weak correlation between the levels of neutralization and the binding activities to viral particles. Our data confirmed that natural CMV infection in healthy women induces potent neutralizing antibodies against infection of both fibroblasts and epithelial cells. The serum neutralizing activities were maintained at high levels throughout the child bearing age. The corresponding titers may serve as a biomarker for CMV vaccine efficacy.

• A novel high-throughput neutralization assay for supporting clinical evaluations of human cytomegalovirus vaccines.

  Authors: Aimin Tang, Fengsheng Li, Daniel C Freed, Adam C Finnefrock, Danilo R Casimiro, Dai Wang, Tong-Ming Fu

  Vaccine. 09/2011; 29(46):8350-6.

  Neutralizing antibodies are considered an important component of protective immunity against congenital infection of human cytomegalovirus (HCMV), a frequently cited cause of birth defects. AnNeutralizing antibodies are considered an important component of protective immunity against congenital infection of human cytomegalovirus (HCMV), a frequently cited cause of birth defects. An effective HCMV vaccine is desired to induce potent neutralizing antibodies in seronegative females, so that the viral transmission to fetus would be blocked if the women contracted HCMV infections during their pregnancies. We describe a novel microneutralization assay to measure antiviral activities against HCMV in serum samples. The assay is based on detection of a dominant HCMV antigen expressed in cells, using near infrared dye-labeled immune reagents. Since the detection is independent of viral cytopathic effects, this assay format has the appeal of a short turn-around time and a read-out that is not subject to operator experience and judgment, making it a promising platform to support large scale clinical studies. In a serological survey of a cohort of 200 healthy females, we showed that the neutralizing titers measured in this assay are highly comparable to those from a neutralization assay based on an enzyme-linked immunostaining method. Lastly, to demonstrate the utility of this assay to support HCMV vaccine study, we presented the results of neutralizing titers from a rhesus macaque vaccination study.

• 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.

• Host genetic determinants of T cell responses to the MRKAd5 HIV-1 gag/pol/nef vaccine in the step trial.

  Authors: Jacques Fellay, Nicole Frahm, Kevin V Shianna, Elizabeth T Cirulli, Danilo R Casimiro, Michael N Robertson, Barton F Haynes, Daniel E Geraghty, M Juliana McElrath, David B Goldstein

  The Journal of infectious diseases. 03/2011; 203(6):773-9.

  Understanding how human genetic variation impacts individual response to immunogens is fundamental for rational vaccine development. To explore host mechanisms involved in cellular immune responsesUnderstanding how human genetic variation impacts individual response to immunogens is fundamental for rational vaccine development. To explore host mechanisms involved in cellular immune responses to the MRKAd5 human immunodeficiency virus type 1 (HIV-1) gag/pol/nef vaccine tested in the Step trial, we performed a genome-wide association study of determinants of HIV-specific T cell responses, measured by interferon γ enzyme-linked immunospot assays. No human genetic variant reached genome-wide significance, but polymorphisms located in the major histocompatibility complex (MHC) region showed the strongest association with response to the HIV-1 Gag protein: HLA-B alleles known to be associated with differences in HIV-1 control were responsible for these associations. The implication of the same HLA alleles in vaccine-induced cellular immunity and in natural immune control is of relevance for vaccine design. Furthermore, our results demonstrate the importance of considering the host immunogenetic background in the analysis of immune responses to T cell vaccines.

• Genetic impact of vaccination on breakthrough HIV-1 sequences from the STEP trial.

  Authors: Morgane Rolland, Sodsai Tovanabutra, Allan C deCamp, Nicole Frahm, Peter B Gilbert, Eric Sanders-Buell, Laura Heath, Craig A Magaret, Meera Bose, Andrea Bradfield [......] Fusheng Li, Steve G Self, Jerome Kim, Susan Buchbinder, Danilo R Casimiro, Michael N Robertson, Ann Duerr, M Juliana McElrath, Francine E McCutchan, James I Mullins

  Nature medicine. 02/2011; 17(3):366-71.

  We analyzed HIV-1 genome sequences from 68 newly infected volunteers in the STEP HIV-1 vaccine trial. To determine whether the vaccine exerted selective T cell pressure on breakthrough viruses, weWe analyzed HIV-1 genome sequences from 68 newly infected volunteers in the STEP HIV-1 vaccine trial. To determine whether the vaccine exerted selective T cell pressure on breakthrough viruses, we identified potential T cell epitopes in the founder sequences and compared them to epitopes in the vaccine. We found greater distances to the vaccine sequence for sequences from vaccine recipients than from placebo recipients. The most significant signature site distinguishing vaccine from placebo recipients was Gag amino acid 84, a site encompassed by several epitopes contained in the vaccine and restricted by human leukocyte antigen (HLA) alleles common in the study cohort. Moreover, the extended divergence was confined to the vaccine components of the virus (HIV-1 Gag, Pol and Nef) and not found in other HIV-1 proteins. These results represent what is to our knowledge the first evidence of selective pressure from vaccine-induced T cell responses on HIV-1 infection in humans.

• Pre-existing adenovirus immunity modifies a complex mixed Th1 and Th2 cytokine response to an Ad5/HIV-1 vaccine candidate in humans.

  Authors: Samuel O Pine, James G Kublin, Scott M Hammer, Joleen Borgerding, Yunda Huang, Danilo R Casimiro, M Juliana McElrath

  PloS one. 01/2011; 6(4):e18526.

  The results of the recent Step Study highlight a need to clarify the effects of pre-existing natural immunity to a vaccine vector on vaccine-induced T-cell responses. To investigate this interaction,The results of the recent Step Study highlight a need to clarify the effects of pre-existing natural immunity to a vaccine vector on vaccine-induced T-cell responses. To investigate this interaction, we examined the relationship between pre-existing Ad5 immunity and T-cell cytokine response profiles in healthy, HIV-uninfected recipients of MRKAd5 HIV-1 gag vaccine (HVTN 050, ClinicalTrials.gov #NCT00849732). Participants were grouped by baseline Ad5 neutralizing antibody titer as either Ad5-seronegative (titer ≤18; n = 36) or Ad5-seropositive (titer >200; n = 34). Samples from vaccine recipients were analyzed for immune responses to either HIV-1 Gag peptide pools or Ad5 empty vector using an ex vivo assay that measures thirty cytokines in the absence of long-term culture. The overall profiles of cytokine responses to Gag and Ad5 had similar combinations of induced Th1- and Th2-type cytokines, including IFN-γ, IL-2, TNF-α, IP-10, IL-13, and IL-10, although the Ad5-specific responses were uniformly higher than the Gag-specific responses (p<0.0001 for 9 out of 11 significantly expressed analytes). At the peak response time point, PBMC from Ad5-seronegative vaccinees secreted significantly more IP-10 in response to Gag (p = 0.008), and significantly more IP-10 (p = 0.0009), IL-2 (p = 0.006) and IL-10 (p = 0.05) in response to Ad5 empty vector than PBMC from Ad5-seropositive vaccinees. Additionally, similar responses to the Ad5 vector prior to vaccination were observed in almost all subjects, regardless of Ad5 neutralizing antibody status, and the levels of secreted IFN-γ, IL-10, IL-1Ra and GM-CSF were blunted following vaccination. The cytokine response profile of Gag-specific T cells mirrored the Ad5-specific response present in all subjects before vaccination, and included a number of Th1- and Th2-associated cytokines not routinely assessed in current vaccine trials, such as IP-10, IL-10, IL-13, and GM-CSF. Together, these results suggest that vector-specific humoral responses may reduce vaccine-induced T-cell responses by previously undetected mechanisms.

• 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.

• 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.

• 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.

• Vaccination with Ad5 vectors expands Ad5-specific CD8 T cells without altering memory phenotype or functionality.

  Authors: Natalie A Hutnick, Diane G Carnathan, Sheri A Dubey, Kara S Cox, Lisa Kierstead, George Makadonas, Sarah J Ratcliffe, Marcio O Lasaro, Michael N Robertson, Danilo R Casimiro, Hildegund C J Ertl, Michael R Betts

  PloS one. 01/2010; 5(12):e14385.

  Adenoviral (Ad) vaccine vectors represent both a vehicle to present a novel antigen to the immune system as well as restimulation of immune responses against the Ad vector itself. To what degreeAdenoviral (Ad) vaccine vectors represent both a vehicle to present a novel antigen to the immune system as well as restimulation of immune responses against the Ad vector itself. To what degree Ad-specific CD8(+) T cells are restimulated by Ad vector vaccination is unclear, although such knowledge would be important as vector-specific CD8(+) T cell expansion could potentially further limit Ad vaccine efficacy beyond Ad-specific neutralizing antibody alone. Here we addressed this issue by measuring human Adenovirus serotype 5 (Ad5)-specific CD8(+) T cells in recipients of the Merck Ad5 HIV-1 vaccine vector before, during, and after vaccination by multicolor flow cytometry. Ad5-specific CD8(+) T-cells were detectable in 95% of subjects prior to vaccination, and displayed primarily an effector-type functional profile and phenotype. Peripheral blood Ad5-specific CD8(+) T-cell numbers expanded after Ad5-HIV vaccination in all subjects, but differential expansion kinetics were noted in some baseline Ad5-neutralizing antibody (Ad5 nAb) seronegative subjects compared to baseline Ad5 nAb seropositive subjects. However, in neither group did vaccination alter polyfunctionality, mucosal targeting marker expression, or memory phenotype of Ad5-specific CD8(+) T-cells. These data indicate that repeat Ad5-vector administration in humans expands Ad5-specific CD8(+) T-cells without overtly affecting their functional capacity or phenotypic properties. This is a secondary analysis of samples collected during the 016 trial. Results of the Merck 016 trial safety and immunogenicity have been previously published in the journal of clinical infectious diseases [1]. ClinicalTrials.gov NCT00849680[http://www.clinicaltrials.gov/show/NCT00849680].

• 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.

• 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.