Merck Ad5/HIV induces broad innate immune PNAS PLUS activation that predicts CD8+ T-cell responses but is attenuated by preexisting Ad5 immunity Daniel E. Zaka,1, Erica Andersen-Nissenb,1, Eric R. Petersonb, Alicia Satob, M. Kristina Hamiltona, Joleen Borgerdingb, Akshay T. Krishnamurtyb, Joanne T. Changb, Devin J. Adamsb, Tiffany R. Hensleyb, Alexander I. Salterb, Cecilia A. Morganb,c, Ann C. Duerrb,c, Stephen C. De Rosab,c,d, Alan Aderema,e,2,3, and M. Juliana McElrathb,c,f,2,3 aSeattle Biomedical Research Institute, Seattle, WA 98109; bVaccine and Infectious Disease Division and cHIV Vaccine Trials Network, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and dDepartment of Laboratory Medicine, eDepartment of Immunology, and fDepartment of Medicine, University of Washington, Seattle, WA 98195 Edited* by Rafi Ahmed, Emory University, Atlanta, GA, and approved October 16, 2012 (received for review June 5, 2012) To better understand how innate immune responses to vaccination recent sieve analyses provide evidence that vaccine responses exerted can lead to lasting protective immunity, we used a systems ap- selective pressure on infecting HIV-1 strains (6). The MRKAd5/ proach to define immune signatures in humans over 1 wk following HIV vaccine received particular attention when the Step Study MRKAd5/HIV vaccination that predicted subsequent HIV-specificT- analysis revealed that certain vaccine subgroups with baseline Ad5 cell responses. Within 24 h, striking increases in peripheral blood seropositivity exhibited increased HIV-1 acquisition rates, halting mononuclear cell gene expression associated with inflammation, its further use in all HIV-1 vaccine trials involving Ad5 seroposi- IFN response, and myeloid cell trafficking occurred, and lympho- tive subjects. Although hypotheses have been generated that may cyte-specific transcripts decreased. These alterations were corrobo- explain vaccine-induced increased HIV-1 infection rates (3, 7, 8) rated by marked serum inflammatory cytokine elevations and and enhanced acquisition was recently recapitulated in the simian immunovirus (SIV) challenge model (9), no clear mechanisms egress of circulating lymphocytes. Responses of vaccinees with pre- fi fi existing adenovirus serotype 5 (Ad5) neutralizing antibodies were have been identi ed to date. These ndings, coupled with the importance of the Ad5 and other adenovirus serotype vectors to strongly attenuated, suggesting that enhanced HIV acquisition in SYSTEMS BIOLOGY vaccine development against many other pathogens (10, 11), Ad5-seropositive subgroups in the Step Study may relate to the lack reinforced our motivation to use an unbiased systems biology of appropriate innate activation rather than to increased systemic approach to better understand the innate immune response trig- immune activation. Importantly, patterns of chemoattractant cyto- gered by MRKAd5/HIV. kine responses at 24 h and alterations in 209 peripheral blood mono- Systems biology integrates global molecular measurements and nuclear cell transcripts at 72 h were predictive of subsequent in- computational analysis with prior knowledge to generate holistic fi + duction and magnitude of HIV-speci c CD8 T-cell responses. This biological insights. This approach therefore provides a framework systems approach provides a framework to compare innate re- to address complex vaccine-induced immunological responses sponses induced by vectors, as shown here by contrasting the more (12, 13). Crosstalk and feedback can be elucidated between im- rapid, robust response to MRKAd5/HIV with that to yellow fever mune signaling pathways and gene regulatory networks operating vaccine. When applied iteratively, the findings may permit selection on multiple spatial and temporal scales. We have previously ap- of HIV vaccine candidates eliciting innate immune response profiles plied systems analysis to identify gene and signaling networks that more likely to drive HIV protective immunity. coordinately amplify and attenuate Toll-like receptor (TLR)- mediated responses underlying innate immune cell activation immunology | innate immunity | systems biology | systems vaccinology | (14–17). Recent systems analyses of responses to vaccination with immunogenicity the highly efficacious YF-17D yellow fever vaccine (18, 19) and seasonal influenza vaccine (20) have yielded novel insights about highly efficacious HIV vaccine offers the greatest promise to their mechanisms of action. Building on this systems-level ap- Ahalt the HIV pandemic. Results of the RV144 study conducted proach, we describe here the innate immune responses induced by in Thailand, where a canarypox vector prime and subunit protein MRKAd5/HIV, how they are impacted by preexisting Ad5 neu- boost regimen showed 31% efficacy for reducing HIV-1 acquisition tralizing antibodies (nAb), how they relate to induction of T-cell (1), have given hope that development of a successful HIV vaccine responses, and how they differ from those induced by live- is possible, and suggest that the vector prime is important for attenuated YF-17D. shaping a protective response. Innate immune responses direct the adaptive immune response and thus influence the potential for in- ducing long-lived protective immunity (2). A comprehensive un- derstanding of the molecular programs underlying optimal innate Author contributions: D.E.Z., E.A.-N., A.A., and M.J.M. designed research; D.E.Z., E.A.-N., responses would therefore facilitate enhanced vaccine design. Little E.R.P., A.S., M.K.H., A.T.K., J.T.C., D.J.A., T.R.H., A.I.S., and M.J.M. performed research; D.E.Z., is known at present about the innate immune responses induced by E.A.-N., E.R.P., A.S., M.K.H., J.B., A.T.K., J.T.C., D.J.A., T.R.H., A.I.S., S.C.D., and M.J.M. ana- candidate HIV vaccines, how these responses drive adaptive im- lyzed data; D.E.Z., E.A.-N., A.A., and M.J.M. wrote the paper; D.E.Z. performed microarray munity, and how these innate responses compare with those in- data analysis; and C.A.M., A.C.D., and M.J.M. implemented the clinical protocol. duced by licensed efficacious vaccines against other pathogens. The authors declare no conflict of interest. To begin to fill these gaps in our knowledge, we conducted a phase *This Direct Submission article had a prearranged editor. Ib clinical trial (HVTN 071) to analyze, at the systems level, human Data deposition: The data reported in this paper have been deposited in the Gene Ex- innate immune responses to the replication-incompetent Merck pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE22822). adenovirus serotype 5 vaccine vector containing HIV-1 inserts gag/ 1D.E.Z. and E.A.-N. contributed equally to this work. pol/nef (MRKAd5/HIV), in parallel with two phase IIb efficacy trials 2A.A. and M.J.M. contributed equally to this work. being conducted using the same vaccine. Although this vaccine did 3To whom correspondence may be addressed. E-mail: [email protected] or not offer protection from HIV acquisition or lower viral loads in the [email protected]. phase IIb Step or Phambili studies (HVTN 502 and 503), it elicited This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. + high CD8 T-cell response rates to the HIV-1 inserts (3–5), and 1073/pnas.1208972109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1208972109 PNAS Early Edition | 1of10 Downloaded by guest on September 24, 2021 Results to MCP-1 and other chemokines (25). Taken together, these MRKAd5/HIV Dramatically Remodels Peripheral Blood Mononuclear results validate the robust systemic innate immune response to fi Cell Transcriptomes by Triggering Robust Innate Immune and Cell MRKAd5/HIV revealed by the transcriptional pro ling. Trafficking Responses. We assessed the innate immune response to MRKAd5/HIV by profiling transcriptomes of peripheral blood The In Vivo Innate Immune Response to MRKAd5/HIV Is Recapitulated mononuclear cells (PBMC) isolated from seven Ad5 nAb sero- in Vitro and Engages a Coordinately Regulated Interacting Network negative individuals (Ad5 nAb titer ≤18; Ad5Neg) during the first Involving Unique Gene Isoforms. To decouple the in vivo innate week after vaccination, by gene-level analysis of Affymetrix exon responses intrinsic to the circulating cells from those associated microarrays. Responses to MRKAd5/HIV peaked at 24 h, with with cells trafficking into and out of the circulation, we extended 1,026 genes exhibiting enhanced and 1,048 genes exhibiting re- our transcriptional profiling to PBMC stimulated with the vac- pressed expression levels compared with prevaccination (Fig. 1A cine vector in vitro. We profiled RNA from unstimulated PBMC and Dataset S1, tab 1). At 72 h postvaccination, the differentially and PBMC incubated for 24 h with MRKAd5 at a dose sufficient expressed genes were a small subset of those detected at 24 h to induce robust cytokine responses (Fig. S1). We found that 8 of (Dataset S1, tab 2). No significantly differentially expressed genes 13 (62%) modules induced in vivo were also induced in vitro and were detected at 168 h. these consisted of the three “Interferon response modules” as We used a modular analysis framework (21) to interpret the well as unannotated modules largely comprised of innate im- transcriptional response. This approach deconvolutes complex mune response genes (Fig. 2A). Remarkably, 92% concordance transcriptional profiles into functionally interpretable patterns between the in vivo and in vitro induction of IFN response genes through the evaluation of combined