T-Cell Responses but Is Attenuated by Preexisting Ad5 Immunity

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 inducing long-lived protective immunity (2). A comprehensive understanding 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 expression responses of pre- was observed (Dataset S1, tab 8). Many of the modules discor- defined disease, cell type, and stimulus-specific coexpressed gene dant between the in vitro and in vivo responses were associated groups. We used versions of the functional modules defined by with particular cellular lineages (myeloid, lymphoid, T cell, B Chaussabel et al. (21, 22) that were updated through meta-analysis cell) or cell-type specific attributes (cytotoxicity) (Fig. 2B), sug- of a much larger transcriptional dataset encompassing many more gesting that the much of the discrepancy between the in vivo and disease states (23), to annotate the differentially expressed gene in vitro responses arose from an absence of cell trafficking in lists and to examine the differential expression of the overall vitro. Comparison with cell-type specific genes lists (20) in- modules themselves. We confirmed the functional annotations of dicated 35% of the genes up-regulated in vivo but not in vitro are the gene modules themselves by performing canonical pathway preferentially expressed in monocytes (Dataset S1, tab 8), sup- enrichment analysis (Dataset S1, tab 3). Mirroring the gene-level porting this hypothesis. results, the modular response peaked at 24 h (13 up-regulated and Our exon-level transcriptional analyses from previous studies 11 down-regulated modules), waned by 72 h (two up-regulated demonstrated that defective alternative mRNA splicing results in modules), and returned to baseline by 168 h (Fig. 1B). Modules profound phenotypic differences in memory T cells (26), and that induced by MRKAd5/HIV were associated with cell intrinsic in- alternative exon use occurs in the innate response. We therefore nate immune responses (“Inflammation” and “Interferon re- extended our analysis of the MRKAd5-induced innate response to sponse” modules) and influx of inflammatory cells (“Myeloid the exon-level to further enrich our understanding of the action of lineage” module). Concomitantly, the “Lymphoid lineage,”“T the vaccine, with the primary focus of identifying vaccine-regulated cells,” and “Cytotoxicity” modules were suppressed, leading to the genes not already detected by the gene-level analysis, particularly hypothesis that the vaccine was stimulating an influx of myeloid those behaving concordantly in vitro and in vivo. Exon-level cells and an efflux of lymphoid cells from the circulation. This analysis led to the identification of 94 additional vaccine-induced hypothesis was further supported by comparing the lists of up- and genes in vivo and in vitro (Dataset S1, tab 9), including critical down-regulated genes with published cell-type enriched gene lists innate immune pathway genes (TLR3, RIPK1,andNLRC5)and generated from meta-analysis of a compendium of sorted cell several genes with important roles in HIV infection (APOBEC3G, transcriptomes (20). Thirty-two percent of the genes we detected APOBEC3F, CCR5,andCD74). Additionally, alternative tran- as up-regulated at 24 h were identified as preferentially expressed scription analysis identified 16 genes with vaccine-induced re- in monocytes in that study, whereas 28% of the down-regulated sponses that varied strongly from exon to exon, but were nev- genes we detected were preferentially expressed in lymphocytes ertheless consistent in vitro and in vivo, including FANCA, FARP2, (Dataset S1, tab 1). Rapid lymphocyte trafficking in response to RERE, GBP6,andGBP7 (Fig. 2C and Dataset S1, tab 10). Al- MRKAd5/HIV is consistent with similar observations made in though the IFN-γ–induced antimicrobial GTPases GBP6 and previous studies with an adenoviral-vectored vaccine (24). Direct GBP7 have been associated with immune responses, most of the canonical pathway enrichment analysis of the regulated gene sets other 16 genes have not been, suggesting additional leads that provided additional support for the module analysis results, in- could be investigated to further understand vaccine-induced im- dicating that innate immune pathways and cell types were up- munological memory. Induction of the unique short isoform of regulated in response to vaccination, and lymphocyte cell types FANCA as part of the MRKAd5-induced innate immune re- and pathways were down-regulated (enrichment results and sponse, for example, provides a compelling link between DNA pathway figures in Dataset S1, tabs 4 and 5). damage pathways and the immunogenicity of adenoviral vectors. We validated the microarray results at the transcript, protein, We performed interaction network analysis to determine and cellular levels. First, we quantified and confirmed the dif- whether the genes commonly regulated by MRKAd5 in vitro ferential expression of mRNAs associated with several vaccine- and in vivo constituted established pathways or represented regulated modules, including “Interferon response” [C-X-C motif isolated nodes. This analysis revealed a densely intercon- chemokine 10 (CXCL10), ISG-15, and STAT1] (Fig. 1C). Next, nected network involving multiple modules and included we corroborated the differential expression of many cytokines genes detected by gene-level analysis, exon-level analysis, and and chemokines at the protein level using multiplex serum alternative transcription analysis [visualized using Cytoscape analyte analysis (Fig. 1D and Dataset S1, tab 6), detecting robust (27) in Fig. 2D]. These findings indicate coordinate regulation changes in serum levels of IP-10, I-TAC, monocyte chemo- of large functional subnetworks, including viral nucleic acid attractant protein-1 (MCP-1), and MCP-2, as well as immuno- sensors, innate immune adaptors, inflammasome components, regulatory IL-10 and IL-1Ra. Finally, we validated the cellular and antiviral effectors. trafficking responses predicted from the modular analysis by directly assessing circulating peripheral blood leukocyte con- Preexisting Neutralizing Antibodies to Ad5 Attenuate the Innate centrations (Fig. 1E and Dataset S1, tab 7), confirming vaccine- Immune Response to MRKAd5/HIV. An important observation in induced influx of monocytes and pronounced efflux of lympho- the Step Study was that the presence of Ad5 nAb before vacci- cyte populations (T, B, and NK cells). Monocyte increases are nation resulted in increased postvaccination risk of HIV acqui- likely a result of recruitment from the bone marrow in response sition (4), and thus far, no clear mechanism for this has been

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Fig. 1. Systems analysis identiﬁes widespread innate immune activation and cellular trafﬁcking responses response to MRKAd5/HIV vaccination in humans. (A) In vivo PBMC transcriptional responses to vaccination with MRKAd5/HIV [n = 7 Ad5 seronegative individuals, false-discovery rate (FDR) ≤ 10%, absolute average

log2 fold-change ≥ 0.5]. Genes significantly differentially expressed in response to MRKAd5/HIV vaccination at any time point are annotated and grouped according to membership in functional gene modules (21, 64). Each column represents subject-specificlog2(fold-changes) compared with prevaccination. To emphasize regulation patterns, expression fold-changes for each gene are scaled by the maximum observed expression response. Pink intensity indicates up- regulation compared with prevaccination, cyan indicates down-regulation. (B) Functional gene modules significantly differentially expressed in PBMC in response

to vaccination. Each black line represents the average log2(fold change) of all genes in the module for a single subject. Up-regulated modules have values >0, down-regulated modules have values <0. FDR < 10%, |average log2(fold-change)|>0.5. (C) Quantitative RT-PCR validation of the differential expression of module-associated genes identiﬁed by microarray analysis. Each line represents the response of one individual (n =7).(D) Protein-level validation of vaccine- induced cytokine and chemokine differential expression by multiplex analyte analysis. (E) Validation of vaccine-induced cellular ﬂuxes predicted by module-level analysis of the PBMC transcriptional responses. *P < 0.05 with Hochberg adjustment from the statistical model assessing change in concentration over time.

elucidated (3, 7, 8). We therefore analyzed the in vivo innate possibility of threshold effects of Ad5 nAb (3, 4), we compared immune responses of vaccinated Ad5 seropositive subjects to the responses between subjects with Ad5 nAb titers ≤ 200 and determine if we could identify alternate programs of innate ac- >200, and thereby identiﬁed 306 seropositivity effect genes (302 tivation in these individuals. The early termination of the Step at 24 h, six at 72 h) for which the vaccine-induced responses were and Phambili studies resulted in the cessation of HVTN 071, markedly attenuated (Dataset S1, tab 11). Canonical pathway limiting the number of subjects we could analyze. Given the enrichment analysis of these genes revealed that induction of

Zak et al. PNAS Early Edition | 3of10 Downloaded by guest on September 24, 2021 Fig. 2. Responses to MRKAd5/HIV are recapitulated in vitro and involve coordinate regulation of an innate immune network involving unique gene isoforms. (A

and B) In vitro responses of modules regulated by MRKAd5/HIV in vivo. Each black line represents the average log2 fold-change of all genes in the module for PBMC derived from separate donors and stimulated in vitro (n = 4); red line indicates average module-level response in vivo. *P ≤ 0.01 comparing module fold-changes observed in vitro those observed in vivo. (A) Gene modules consistently regulated by MRKAd5 in vivo and in vitro. (B) Gene modules regulated in vivo but not in vitro. (C) Single gene heatmaps showing average exon-level expression responses to MRKAd5/HIV in vivo (n = 7) and in vitro (n = 4). Pink intensity indicates up-regulation, cyan indicates down-regulation. (D) Innate immune response interaction network of genes consistently induced in response to MRKAd5 in vivo and in vitro, arranged by representative subcellular localization. Node colors indicate functional module associations; node shapes indicate the mode of differential expression. Red lines indicate protein-protein interactions, gray lines indicate mutual membership in common complexes, and blue edges indicate protein–DNA interactions.

complement pathways, innate immune sensors, and G-protein cytokine concentrations, measured 24 h postvaccination (Fig. 1D coupled receptor signaling was significantly attenuated (Dataset and Dataset S1, tab 6), could predict the Gag-specificCD8+ T- S1, tab 12). This attenuation extended to all modules regulated by cell response magnitudes. We performed two analyses: (i)dis- the MRKAd5 vaccine (Fig. 3A), including impaired down-regula- crimination between subjects with detectible (CD8pos =CD8mod tion of lymphocyte modules and genes preferentially expressed in and CD8high) and undetectable (CD8neg) responses; and (ii)dis- lymphocytes (Dataset S1, tab 11), suggesting suppression of the crimination between subjects with high (CD8high) and moderate acute lymphopenia observed in the Ad5 seronegative subjects (Fig. or undetectable (CD8mod,CD8neg) responses. The predictive 1E). Direct comparison between regulation of innate immune potential of individual cytokines and all cytokine pairs was eval- networks in seronegative and Ad5 nAb >200 subjects revealed uated by 60 iterations of eightfold cross-validation of linear dis- coordinate dysregulation that included the RIG-I, NLR/inflam- criminant analysis (LDA) classifiers. masome, and TLR pathways (visualized using Cytoscape in Fig. Two chemokines, MCP-1 and MCP-2 (Fig. S4 A and B), dis- 3B). Finally, we validated these transcriptional results at the pro- criminated between CD8mod/CD8high subjects and CD8neg subjects tein level by analyzing serum analytes from a larger set of vacci- with high accuracy (81% and 88%, respectively), and thus were nated subjects (Fig. S2). Consistent with the transcriptional results, qualitatively predictive of the vaccine CD8+ T-cell immunoge- cytokine responses were markedly attenuated in Ad5 nAb >200 nicity. In both cases, higher chemokine induction predicted in- subjects compared with Ad5 nAb ≤ 200 subjects (Fig. 3C and creasedlikelihoodofpositiveCD8+ T-cell responses. Combining Dataset S1, tab 13). Taken together, these results suggest that the MCP-1 and MCP-2 into a single classifier did not increase pre- predominant effect of preexisting Ad5 nAb on the innate immune dictive accuracy. However, the accuracy was increased by combi- response is global attenuation. These data do not support the nation with other cytokines that were not individually predictive hypothesis that preexisting immunity leads to enhanced systemic (Fig. 4A and Fig. S4A). For example, the growth factor PDGF-AA innate immune activation. was 71% predictive individually but 85% predictive in combination with MCP-1. The network of predictive pairwise signatures MRKAd5/HIV Innate Immune Responses Predict Immunogenicity. We for CD8+ T-cell responses is shown in Fig. 4A, and the receiver next identified MRKAd5/HIV-induced innate immune sig- operating characteristic (ROC) for predicting positive CD8+ T- natures that predict subsequent HIV-specific adaptive immune cell responses based on GRO and MCP-2 is shown in Fig. 4B. responses. Based on the frequency of Gag-specific CD8+ T-cell Classifiers performing as well as MCP-2 individually (or top pairs responses detected at day 28 after one immunization (Fig. S3), involving MCP-2) were generated only 13% of the time when the we categorized vaccine recipients (n = 31) into high, moderate, analysis was repeated on randomized datasets, indicating that the or low responders. We determined whether fold-changes in serum result is moderately robust. Nevertheless, repeat analyses using

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Fig. 3. Preexisting Ad5 neutralizing antibodies attenuate the MRKAd5/HIV-induced innate immune response in vivo. (A) Effect of Ad5 nAbs on gene modules regulated by MRKAd5/HIV. Black line represents the average fold-change of all genes in the modules, averaged over 8 Ad5 nAb ≤ 200 subjects. Green lines represent the average fold-change of all genes in the module for the two Ad5 nAb > 200 subjects. (B) Attenuated induction of critical innate immunity pathways in Ad5 nAb > 200 compared with Ad5 nAb ≤ 200 subjects. Nodes are colored according to average extent of induction in Ad5 nAb ≤ 200 subjects at 24 h (Left) and average extent of response attenuation in Ad5 nAb > 200 subjects compared with Ad5 nAb ≤ 200 subjects (Right). Edges represent protein–protein interactions between nodes obtained from InnateDB (65). (C) Representative attenuation of MRKAd5/HIV serum cytokine induction in Ad5 nAb > 200 subjects. Se- rum IP-10/CXCL10 concentrations are contrasted between Ad5 nAb ≤ 200 (n = 27) and Ad5 nAb > 200 (n = 6) individuals. Shading represents the inter- quartile ranges with the 75th percentile shown on top and the 25th percentile shown below the median line. *P < 0.05 after Hochberg adjustment.

similar vaccines are required to confirm the association between strategy enhanced vaccine-induced T-cell responses (30). Because these chemokines and CD8+ responses. In the second analysis, the interaction network analysis of the overall CD8+ T-cell response SYSTEMS BIOLOGY combination of RANTES (regulated upon activation, normal T gene set itself was found to be uninformative, we investigated cell expressed and secreted) and IL-28A was predictive of CD8+ whether any the gene set members are protein–protein interaction response magnitudes with high accuracy (87%), even though neighbors of genes belonging to MRKAd5 regulated functional neither cytokine was strongly predictive individually (Fig. S4 C and modules (Fig. 1B). We found that many of the CD8+ T-cell re- D). Strong down-regulation of RANTES or up-regulation of IL- sponse associated genes are nearest neighbors of members of the 28A was associated with induction of high magnitude CD8+ T-cell “Cytotoxicity,”“T cells,” and “Lymphoid lineage” modules (Fig. responses (Fig. S4D). The ROC for predicting high magnitude 5C), providing additional support for the association between CD8+ T-cell responses based on IL-28A and RANTES is shown these genes and CD8+ T-cell immunogenicity. in Fig. 4C. Repeating the analysis on randomized datasets generated classifiers performing as well as IL-28A and RANTES 25% Replication-Incompetent MRKAd5 Induces a Greater Number of of the time, indicating that this particular result should be regar- Innate Immune Genes than Does Live-Attenuated YF-17D, but the ded as a hypothesis until additional studies have validated the role Response Is More Transient. Recent studies have suggested that of these cytokines in the fine tuning of adenoviral vector CD8+ T- the efficacy of live-attenuated YF-17D, a yellow fever vaccine (31), cell immunogenicity. may result from robust innate immune activation (18, 19, 32). We therefore contrasted the transcriptional responses induced Using Systems Biology to Generate Additional Hypotheses Regarding MRKAd5 and published in vivo profiles for YF-17D (19). Al- the Immunogenicity of MRKAd5/HIV. To identify additional potential though the MRKAd5/HIV vaccine induced more than 1,000 genes mechanisms controlling MRKAd5/HIV-induced T-cell responses, and repressed a similar number, the YF-17D vaccine only induced we extended our signatures analysis to the transcriptional level. 181 genes and repressed 10 genes (Fig. 6A). However, the re- Given our small data sample sizes, it was not possible to imple- sponse to MRKAd5/HIV was rapid and transient, but the re- ment approaches described above or in previous studies (19, 20), sponse to YF-17D lagged and was persistent (Fig. 6A). Modular and the results must be regarded as hypothesis-generating until analysis further illuminated differences between the two vaccines clinical studies using similar vaccines make validation of the results (Fig. 6B). Whereas MRKAd5/HIV induced the “Inflammation,” possible. We defined groups of subjects with high-, moderate-, or “Interferon response,” and “Myeloid lineage” modules and low-magnitude CD8+ T-cell responses by 1D clustering of the gag- inhibited the “Lymphoid lineage,”“T cells,” and “Cytotoxicity” specific responses. Genes with statistically significant differences modules (Figs. 1B and 4B), YF-17D vaccination induced only in vaccine-induced transcriptional responses between the high and a subset of the “Interferon response” modules (M1.2 and M3.4) low CD8+ groups were then identified through direct comparison. (Fig. 6B). Surprisingly, significant differences between these groups of sub- Given that dosage and replication kinetics could likely account jects were only identified from the transcriptional responses for the gross differences in innate immune activation between measured 72 h postvaccination (88 genes were positively associ- replication defective MRKAd5 and live-attenuated YF-17D, we ated and 121 genes were negatively associated) (Fig. 5 A and B and performed a new set of comparative in vitro experiments to di- Dataset S1, tab 14), and none of the MRKAd5-responsive mod- rectly contrast responses to the two vaccines, focusing first on ules differed significantly between the groups. Several of the im- differences identified in vivo that recapitulated in vitro. We plicated genes have clear functional relationships to cytotoxic identified 43 genes preferentially induced by MRKAd5/HIV in responses, including the inhibitory killer cell Ig-like receptor vivo that confirmed in vitro (Fig. 6C), including several associ- KIR2DL1, the NK-cell activating receptor CLEC2D (28), and the ated with innate immune responses (IRF1), the complement NK-cell signaling adaptor EWS-FLI1–activated transcript 2 (EAT- pathway (C1QB), pathogen recognition (TLR8), the inflamma- 2) (29) (Fig. 5 A and B). Consistent with this association between some (CASP10, P2RX7), and NK-cell activation [SLAMF7 (33)]. EAT-2 expression and CD8+ T-cell responses, it was recently This group also included several immunosuppressive factors, reported that adenoviral expression of EAT-2 as part of a vaccine including the T-cell inhibiting IDO1 (34), M2-macrophage

Zak et al. PNAS Early Edition | 5of10 Downloaded by guest on September 24, 2021 scription factors (Fig. 6D), and that these transcription factors potentially coregulate each other. Although there were no genes preferentially induced by YF-17D in vivo that validated in vitro, we identiﬁed a robust gene set, predominantly consisting of

Fig. 4. Vaccine-induced serum cytokine and chemokine responses predict the magnitudes of CD8+ T-cell responses induced by MRKAd5/HIV. (A) Network depiction of serum cytokines and chemokines with 24-h vaccine-induced fold- changes that discriminate subjects who develop CD8+ T-cell responses (28 d after vaccination) from those who do not. Node sizes indicate predictive accuracy of the factors individually (percentages inside nodes), node color, and intensity indicate positive (pink) or negative (blue) correlations between induction levels of the factor and CD8+ T-cell response magnitudes. The presence of an edge between nodes indicates that the two cytokines in combination have increased predictive accuracy compared with either cytokine alone. Edge width is proportional to predictive accuracy of the pair-wise signature of the connected nodes (red percentages). (B) ROC for predicting which subjects will develop CD8+ T-cell responses, based on 24-h vaccine induced fold-changes of GRO and MCP-2 in a LDA model. The red line indicates the tradeoff in false positive rate required as the true positive rate is increased. Prediction accuracies were estimated from over 60 rounds of Fig. 5. Systems analysis identiﬁes innate immune response genes that are eightfold cross-validation. AUC, area under the curve. (C) ROC for predicting + associated with the immunogenicity of MRKAd5/HIV. (A) The 209 genes with which subjects will develop high magnitude CD8 T-cell responses, based on 72-h MRKAd5/HIV-induced expression responses that are positively (Upper) 24-h vaccine induced fold-changes of IL-28A and RANTES in a LDA model. or negatively (Lower) associated with the magnitude of MRKAd5/HIV-in- Notation is as in B. duced CD8+ T-cell responses. Subjects with low and high magnitude CD8+ T-cell responses are on the Left and Right halves of the heatmap, respectively.

Gene selection criteria: FDR ≤ 20% and |average log2(fold-change)| ≥ 0.5. skewing TF KLF4 (35), macrophage inhibitor PSTPIP2 (36), and Rows are scaled as in Fig. 1A. Pink intensity indicates up-regulation compared the PD-1 death receptor ligand PDCD1LG2. The preferential with prevaccination, cyan indicates down-regulation. (B) Seventy-two hour induction of three transcription factors by MRKAd5, IRF1, MRKAd5/HIV-induced expression responses of two representative genes, + KLF4,andSTAT5A suggested that these factors may partly stratified by the magnitude of the CD8 T-cell responses observed in the same subjects. (C) Protein–protein interaction network emphasizing links between account for the PBMC transcriptome induced by MRKAd5. By + fi – fi CD8 T-cell response-associated genes identi ed in Fig. 5A (triangles) and mining published ChIP-Seq datasets (37 39), we con rmed that constituents of functional gene modules differentially regulated by MRKAd5/ several MRKAd5-specific genes are direct targets of these tran- HIV (circles), colored according to module associations.

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Fig. 6. MRKAd5 induces more extensive innate immune activation than the gold-standard yellow fever vaccine, YF-17D. (A) MRKAd5/HIV and YF-17D trigger innate immune responses in PBMC with distinct kinetics in vivo. The number of genes significantly up-regulated (upper half) or down-regulated (lower half) in response to vaccination with MRKAd5/HIV (red, present study) or YF-17D [blue (19)] are plotted. Significant differential expression defined as FDR ≤ 10%, |

average[log2(fold-change)]|>0.5; n = 7 (MRKAd5/HIV), n = 25 (YF-17D). (B) MRKAd5/HIV regulates a broader spectrum of gene modules in vivo than does YF- 17D. Each line represents the average fold-change of all genes in a module for a given individual (red lines, MRKAd5/HIV vaccines; blue lines, YF-17D vaccinees). Modules labeled in red differ signiﬁcantly between MRKAd5/HIV and YF-17D in vivo (FDR ≤ 10%), modules labeled with asterisks differ significantly between MRKAd5 and YF-17D in vivo and in vitro (FDR ≤ 10%). (C) Average expression responses in vivo and in vitro of 43 genes preferentially induced by MRKAd5. Genes associated with functional gene modules are indicated. Rows are scaled as in Fig. 1A. Pink intensity indicates up-regulation compared with

controls, cyan indicates down-regulation. FDR < 10%, |average log2(fold-change)|>0.5 for induction in response to MRKAd5 and comparing MRKAd5 to YF- 17D, in vivo and in vitro. (D) Putative transcriptional regulatory network controlling innate immune responses preferentially induced by MRKAd5. Squares indicate transcription factors preferentially induced by MRKAd5 in vivo and in vitro; circles indicate target genes preferentially induced (pink) or repressed (light blue) by MRKAd5 in vivo and in vitro. Lines indicate protein-DNA transcription factor–target gene interactions identiﬁed from published ChIP-seq datasets [blue, KLF4 targets (38); purple, IRF1 targets (37); orange, STAT5A targets (39)]. Pink edges indicate IRF1 target genes identiﬁed by conventional methods (66–68). (E) Expression responses of CRIP3 and NPB (72–168 h postvaccination) are negatively associated with CD8+ T-cell response magnitudes

induced by MRKAd5/HIV and YF-17D. Shown are log2(fold-changes) compared with prevaccination of the two genes for both vaccines, stratiﬁed by the magnitudes of vaccine-induced CD8+ T-cell responses observed in the same subjects. Lines indicate mean values.

members of the “Interferon response” modules (including STAT1, spectively, compared with 226 genes being robustly induced in STAT2, IRF7, and IFI27), that was induced by both vaccines in common (Dataset S1, tab 16). Similar differences in the down- vitro and in vivo (Dataset S1, tab 15). regulated gene sets were observed, with 190 and 229 genes being To generate hypotheses about differences in innate immune preferentially down-regulated by MRKAd5 and YF-17D, re- activation that may result at the actual sites of MRKAd5 or YF- spectively, compared with 137 genes being down-regulated in 17D vaccination, we also performed a direct comparison between common. MRKAd5 preferentially induced T-cell chemoattractants the in vitro responses to the two vaccines, without constraining (CXCL9/10/11), MHC genes, and T-cell–associated cytokines them by the in vivo results. Unexpectedly, the innate activation (IFNG, IL-2,andIL-7) but YF-17D preferentially induced the proﬁles of MRKAd5 and YF-17D differed more strongly in vitro IFNA family of antiviral cytokines and several neutrophil chemo- than we had originally observed in vivo, with 349 and 313 genes kines (IL-8, CXCL2, -3, -5,and-6). Canonical pathway enrichment being preferentially induced by MRKAd5 and YF-17D, reanalysis reinforced the differences between the two vaccines, with

Zak et al. PNAS Early Edition | 7of10 Downloaded by guest on September 24, 2021 MRKAd5-specific gene enrichments including “Antigen Pre- Few vaccine vectors in development match Ad5 in terms of the sentation Pathway” and “T Helper Cell Differentiation” and YF- magnitude and frequency of vaccine insert-specific CD8+ T cells 17D–specific gene enrichments, including “Systemic Lupus Eryth- they induce (Fig. S3) (3). Ad5-induced CD8+ T cells are func- ematosus Signaling” and several IL-17 associated pathways (Dataset tional in some settings, because they are essential to the efficacy S1, tab 17). These results indicate that greater specificity in vaccine- of Ad5-vectored Ebola vaccines in the nonhuman primate model induced innate immune responses may be revealed by profiling local, (10) and also appear to have exerted selective pressure on rather than systemic responses. infecting HIV-1 in the Step Study (6). Results in the murine Finally, we evaluated whether the transcriptional signatures model, however, show that Ad5 induced CD8+ T cells may not + properly differentiate into memory cells required for protective associated with enhanced CD8 T-cell responses induced by † MRKAd5/HIV (Fig. 5A) were also associated with enhanced responses. These contrasting results suggest that although the CD8+ T-cell responses to YF-17D, despite the numerous differ- strong Ad5-induced CD8+ T-cell response may be sufficient for ence between the vaccines. By reanalyzing published YF-17D vaccine efficacy in some systems, increases in the efficacy of Ad5- transcriptome and longitudinal CD8+ T-cell response data (19) based vaccines may be achieved if the quality of the induced + using the approach implemented above, we identified two genes, CD8 T cells is optimized. CRIP3 and NPB, with vaccine-induced expression responses that To determine how the magnitude and quality of vaccine-in- were consistently associated with impaired CD8+ T-cell responses duced T-cell responses are shaped and may ultimately be opti- to both vaccines (Fig. 6E). Strengthening the associations, the mized by activation of innate pathways, we performed two average fold-changes for these genes for moderate CD8+ response hypothesis-generating analyses. First, we evaluated innate im- + mune response signatures that were associated with vaccine-in- subjects was always between that of high and low CD8 response + subjects, even though the data for the moderate subjects was not duced CD8 T-cell magnitude, and second, we compared its fi fi used in the gene selection. innate activation pro le with that of the highly ef cacious yellow Taking these data together, we have defined the early innate fever vaccine YF-17D. immune response to the MRKAd5/HIV vaccine, identified an In the signature analyses, we found that serum induction of the attenuated innate response in individuals with Ad5 nAb, and two chemokines, MCP-2 and MCP-1, 24 h postvaccination, fi predicted whether or not a subject would develop a measureable de ned innate response signatures that predict CD8+ T-cell + responses to Gag. These data suggest previously unexplored CD8 T-cell response 4 wk postvaccination (Fig. 4 A and B, and Fig. S4 A and B). Predictive accuracy was increased to nearly targets for enhancing the immunogenicity of next-generation fl HIV vaccines. 90% by coupling these chemokines with proin ammatory cytokines (Fig. 4 A and B, and Fig. S4 A and B). Although additional studies are required to confirm this result, a role for these che- Discussion + Systems biology analysis can contribute to rational vaccine design mokines in CD8 T-cell responses is supported by the strong T- in four major ways. First, it can enable the identification of cor- cell chemoattractant function they exhibit (47) and the reported CD8+ T-cell adjuvant activity of MCP-1 (48). Furthermore, relates of immunogenicity and protection; second, it can reveal the there was an indication in our data that subjects with the highest regulatory networks within cells that lead to the desired host im- CD8+ T-cell responses were those who either strongly down- mune response; third, it can guide the reengineering of the vaccine regulated RANTES or up-regulated IL-28A (Fig. 4C,and Fig. S4 regimen to favor desirable responses; and finally, it can supply C and D). One hypothesis is that strong down-regulation of se- tools to glean insight from failed candidate vaccines. We believe rum RANTES postvaccination may indicate increased uptake by this study provides fresh understanding in each of these ways to the fl fi migrating in ammatory cells, but increased serum IL-28A may highly immunogenic but nonef cacious MRKAd5/HIV vaccine. indicate an immunogenic role for the antiviral activities of this Despite inducing T-cell responses at a high frequency, cytokine (49, 50). The transcriptional CD8+ signature analysis MRKAd5/HIV neither reduced HIV-1 acquisition nor lowered also revealed many genes exhibiting responses to MRKAd5/HIV viral loads postacquisition in two independent clinical trials (3, 5). at the 72-h timepoint that were significantly associated with Furthermore, Ad5 seropositive male vaccine recipients in the Step CD8+ T-cell response magnitudes (Fig. 5 A and B), including study showed an increased rate of HIV-1 acquisition, making the several that are nearest neighbors of CD8+ T-cell response-as- influence of Ad5 nAbs on vaccine responses an area of intense – sociated module genes (Fig. 5C). One compelling component of research (3, 4, 7, 8, 40 42). Although additional factors likely the gene signature was EAT-2 (Fig. 5B), which was recently played a role in acquisition (43–45), the effect of Ad5 nAbs was fi reported to enhance the frequency of vaccine induced T cells signi cant and has been supported in the nonhuman primate SIV when encoded in an Ad5 vector (30). Despite the striking dif- challenge model. One current hypothesis is that antibody-medi- ferences in innate response kinetics between the vaccines, we ated internalization of Ad5 results in increased dendritic cell ac- also tested whether the CD8+ signature for MRKAd5/HIV was tivation, which may lead to enhanced HIV-1 infectivity (40). In our associated with CD8+ T-cell response magnitudes for YF-17D. study, we found no evidence for enhanced or prolonged systemic Reanalysis of the published YF-17D dataset (19) identified two innate immune responses in volunteers with preexisting Ad5 nAb. genes, CRIP3 and NPB, with induction patterns that were asso- Instead, we observed attenuation of the overall transcriptional ciated with the CD8+ T-cell responses of both MRKAd5/HIV response (Fig. 3 A–C), which we confirmed at the protein level by and YF-17D (Fig. 6E). Roles for both of these genes in vaccine multiplex serum cytokine analysis (Fig. 3D and Dataset S1, tab 13). mechanisms are plausible, given the function of CRIP3 in thymic Our results are compatible with the suggestion that Ad5 nAb may cellularity (51) and the high expression levels of NPB in lym- effectively lower the dose of the vaccine detected by the innate phoid tissues (52). immune system (8, 46) and are consistent with the reduced vaccine In the comparative analysis, we found a striking difference in immunogenicity seen in vaccine recipients with nAb titers >200 the temporal innate immune activation profile of MRKAd5/HIV (3). Our results are also compatible with a model in which nAbs and YF-17D (Fig. 6A) that is consistent with, but not completely negatively regulate innate signaling pathways. The latter hypoth- explained by, the dosage and pharmacokinetics of the vaccines: esis is of interest given the possibility that the opsonized vector although replication-incompetent MRKAd5/HIV is present at could interact with Fc receptors on antigen presenting cells; an the highest levels immediately after injection (53), live-attenu- event that might result in an inappropriate context for pre- ated YF-17D takes 5–7 d to reach maximal titers in the host (31). sentation of the vaccine-encoded antigens. Regardless of the Unexpectedly, the innate immune response to MRKAd5/HIV precise mechanism, our observations highlight the impact of preexisting type-specific immunity to the vector on vaccine responses † and open new avenues for mechanistic studies into the effects of Sarkar S, et al, Keystone Symposia on Molecular and Cellular Biology, October 27– this important variable. November 1, 2010, Seattle, WA.

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1208972109 Zak et al. Downloaded by guest on September 24, 2021 was much more extensive than that induced by YF-17D (Fig. 6 A vaccination) for 10 subjects (three Ad5-seropositive and seven Ad5-seroneg- PNAS PLUS and B). Although the peak response to MRKAd5/HIV involved ative). For in vitro profiling, eight samples were analyzed: PBMC obtained induction of over a dozen functional modules, the peak response from four Ad5 seronegative donors stimulated for 24 h with MRKAd5 empty to YF-17D involved induction of only two. In vitro stimulation vector at 20,000 particles per cell and GTS buffer mock control. experiments with the two vaccines identified which of the innate immune response differences observed in vivo are because of Agilent 3′ Arrays. RNA from the MRKAd5 vs. YF-17D comparative in vitro study cell-intrinsic differences in innate immune signaling (Fig. 6 C and was analyzed using the Agilent SurePrint G3 Human GE 8 × 60K microarray D). MRKAd5 preferentially induced a transcriptional regulatory platform (Agilent Technologies), essentially as described in ref. 17. For fi network involving three transcription factors, IRF1, KLF4,and comparative in vitro pro ling, 16 samples were analyzed: PBMC obtained STAT5A, in vivo and in vitro (Fig. 6 C and D). Activation of the from four Ad5 seronegative donors stimulated for 24 h with MRKAd5 empty IRF1 network may play a part in the strong CD8+ T-cell immu- vector at 60,000 particles per cell and GTS buffer mock control or YF-17D at nogenicity of MRKAd5/HIV, given the role of this transcription 30 particles per cell and DMEM 2% (vol/vol) FCS buffer mock control. + Microarray data analysis procedures are described in the SI Materials and factor in regulating MHC class I presentation and CD8 T-cell Methods. Quantitative real-time PCR was performed as described in ref. 17. responses (54). Interestingly, a number of the MRKAd5-specific genes are also associated with immunoregulatory functions. Multiplex Cytokine Analysis. Serum cytokine analysis was performed using the Among these, KLF4 promotes anti-inflammatory “M2” and fl “ ” Lincoplex High Sensitivity kit (Millipore Cat# HSCYTO-60SPMX13) and regular inhibits proin ammatory M1 macrophage polarization (35), sensitivity kits (Millipore Cat# MPXHCYTO60KPMX42, MPXHCYP2:00 PMX23, and PDCD1LG2 and IDO1 suppress T-cell activation through and MPXHCYP3-PMX9), according to the manufacturer’s instructions (Linco/ a variety of mechanisms (55, 56). Further study will determine Millipore), and samples were analyzed on a Luminex 200 (Luminex). PBMC whether pharmacological inhibition of these molecules will lead supernatants were assayed similarly for a subset of the analytes. Data were to enhanced Ad5-induced T-cell functionality. Finally, direct analyzed using a custom in-house export and quality control program in comparison between MRKAd5- and YF-17D–induced innate im- conjunction with the Ruminex program (58). mune responses in vitro revealed additional functionally relevant differences between the vaccines, suggesting that profiling of local Enumeration and Phenotyping of Fresh Blood Cell Populations. Trucount tubes responses may complement measurements of systemic responses (BD) were stained with CD45-ΑPC, CD14-PE, CD3-PerCp, and CD8-FITC (all obtained by from blood cell transcriptomes. from BD). For further phenotyping, whole blood was diluted 1:10 in Pharm- Our comprehensive analysis of the immediate systemic re- lyse RBC lysis buffer (BD), incubated 10 min at room temperature and sponse following vaccination with MRKAd5 provides fresh un- centrifuged at 750 × g for 5 min. RBC lysis was repeated and cells were derstanding of vaccine-induced innate immune activation, how it resuspended in cold PBS; 2 × 106 to 6 × 106 cells were stained with Aqua Vi-

is modulated by preexisting immunity, and how it relates to the ability Dye (Invitrogen), followed by one of three antibody mixtures (details SYSTEMS BIOLOGY subsequent adaptive immune responses. Such understanding will provided upon request). Cells were fixed with PBS containing 1% para- play an important role in the development of a highly efficacious formaldehyde and stored at 4 °C until analysis by flow cytometry. All samples HIV vaccine. from one volunteer were analyzed together within 7 d of staining.

Materials and Methods Statistical Analysis of Cell Concentration and Multiplex Cytokine Data. Analysis Subjects. We enrolled 35 healthy HIV-1-uninfected adults [median age 37 y of analytes altered after vaccination was performed by running a mixed (range 20–50); 21 female; 28 Caucasian, 7 African American]. Eleven subjects model with normally distributed errors and an unstructured covariance were Ad5Positive and 24 were Ad5Neg (Fig. S2). Microarrays were run on five matrix, with cytokine or cell concentration as the dependent variable and males and five females [median age 33 y (range 22–43); nine Caucasians]. categorical sampling time as the independent variable, allowing random Female participants were counseled to use birth control and avoid preg- intercepts for each participant for each immunization series and each cy- nancy during the study. All participants provided written informed consent, tokine or cell type. Sex and age were included as possible confounders. P and each of the four United States trial sites obtained approval for the study values for time were adjusted within a given vaccine series using the fi fi through their institutional review boards. Hochberg method (59). Methods for identi cation of serum analyte pro les that were predictive of CD8+ T-cell responses are provided in the SI Materials Study Design. HVTN 071 was a Phase 1b multicenter, open-label trial and Methods. (ClinicalTrials.gov #NCT00486408). At the start of the trial (day 0), all volunteers were intramuscularly vaccinated with 1.5 × 1010 genomes of the previously- In Vitro PBMC Stimulations. One-million PBMC from healthy Ad5-seronegative described MRKAd5/HIV vaccine (3); 24 received a second vaccination at day 28 individuals were stimulated with MRKAd5 empty vector or GTS buffer mock before all MRKAd5/HIV vaccinations were suspended (4). Blood was collected control (60) or YF-17D or DMEM with 2% (vol/vol) FCS mock control [courtesy immediately before vaccination and at 4–6, 24, 72, and 168 h postvaccination of Charles Rice, Rockefeller University, New York (61–63)] in RPMI containing for 11 individuals. Serum was obtained from an additional 24 individuals at the 10% (vol/vol) FCS, penicillin and streptomycin at a range of multiplicities of prevaccination and 24-h time points. infection. After 24 h, cell-culture supernatants were harvested for multiplex cytokine analysis and cells were frozen in RLT buffer (Qiagen) containing β fi Microarrays and Quantitative Real-Time PCR. PBMC were isolated from blood -mercaptoethanol, for RNA puri cation and microarray analysis. as previously described (57). RNA was extracted from PBMC using the RNeasy Protect Cell protocol (Qiagen). Before labeling, the integrity of samples was ACKNOWLEDGMENTS. We thank the HVTN 071 Protocol Team, Michael checked using an Agilent 2100 Bioanalyzer. Robertson, Youyi Fong, Greg Spies, Jennifer Vogt, Jim Simandl, Don Carter, Stephen Voght, Lamar Fleming, Marcus Altfeld, and Galit Alter for their assistance, and the James B. Pendleton Charitable Trust for their generous Affymetrix Exon Arrays. RNA expression for the in vivo study and one in vitro equipment donation. This work was supported by National Institutes of Health study was analyzed using the Human Exon ST 1.0 microarray platform (Affy- Grants UM1 AI068618 and U01 AI069481 (to M.J.M.); the Bill and Melinda metrix) essentially as described in ref. 17. For in vivo profiling, 50 samples were Gates Foundation Collaboration for AIDS Vaccine Discovery Grant 38645 (to analyzed: five time points (prevaccination and 6, 24, 72 and 168 h post- M.J.M.); and National Institutes of Health Grant T32 AI007140 (to E.A.-N.).

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