CombiningDNAVaccineandAIDA-1inAttenuated Salmonella ......Research Article CombiningDNAVaccineandAIDA-1inAttenuated Salmonella Activates Tumor-Speciﬁc CD4þ and CD8þ T-cell Responses

Research Article

CombiningDNAVaccineandAIDA-1 inAttenuatedSalmonella Activates Tumor-Specific CD4þ andCD8þ T-cell ResponsesYu Mei1,2, Lixiang Zhao3, Yonghao Liu1, Huanle Gong1, Yuan Song1, Lei Lei1,Ying Zhu1, Ziqi Jin1, Shoubao Ma1, Bo Hu1, Qing Sun4, and Haiyan Liu2

Abstract

Stimulation of tumor-specific responses in both CD4þ andCD8þ T cells has been a challenge for effective tumor vaccines.Wedesigned a vaccine vector containing the AIDA-1 autotransporterand DNA vaccine elements, generating a murine melanomavaccine that was delivered by the attenuated Salmonella strainSL7207. Growth of murine subcutaneous melanoma was signif-icantly inhibited by intranasal immunization with the Salmonellatumor vaccine. The vaccine activated tumor-specific CD4þ andCD8þ T-cell responses, with increased T-cell proliferation, tumorantigen–specific Th1 cytokine production, increased percentagesof tetramer positive cells, and cytotoxicity. CD4þ or CD8þ T-celldepletion resulted in the loss of antitumor activity of the Salmo-nella tumor vaccine, suggesting that the efficacy of the vaccine was

dependent on both CD4þ and CD8þ T cells. Lung metastasis ofthe tumor was also inhibited by vaccine treatment. Similarly, thepercentages of tumor-specific Th1 cytokine production by CD4þ

and CD8þ T cells in the spleen, tumor, and bronchoalveolarlavage were increased after vaccine treatment. Tumor-specificproliferation of CD4þ and CD8þ T cells was also promoted bythe vaccine. Tetramer staining and cytotoxicity assay showedenhanced tumor-specific CD8þ T-cell response after vaccine treat-ment. Therefore, the Salmonella tumor vaccine could activateboth tumor-specific CD4þ and CD8þ T-cell responses. This vac-cine strategymay be widely applicable to the development of oralor nasal vaccines against tumors. Cancer Immunol Res; 5(6); 503–14.�2017 AACR.

IntroductionInduction of antitumor immune responses in patients is one of

the most sought-after goals in cancer immunotherapy. Generally,most of these therapies are directed toward enhancing the CD8þ

T-cell response, as CD8þ T cells are specialized for lytic function(1, 2). However, CD4þ T cells also play an important role infacilitating the antitumor response. Tumor-specific CD4þ T cells,particularly T-helper type 1 (Th1) cells, are necessary for thepriming of tumor-specific CD8þ T cells, and essential for thegeneration and maintenance of long-lasting cytotoxic T lympho-cyte (CTL) responses (3–10). Therefore, stimulation of both

tumor-specific Th1 and CTL responses is important for the effec-tive antitumor immunotherapy.

To maximize uptake and presentation of vaccine MHC class IIpeptides, proteins can be expressed on the surface of gram-negative bacteria. For example, surface display of antigens onattenuated Salmonella induces a specific CD4þ T-cell response invivo in a Helicobacter pylori vaccine (11). That vaccine took advan-tage of the Adhesin Involved in Diffuse Adherence (AIDA) pro-tein, an autotransporter that has been co-opted frequently forexpression of recombinant protein on the surface of gram-nega-tive bacteria (12–14). AIDA is synthesized with an N-terminalsignal peptide followed by a passenger domain and a C-terminaldomain containing the membrane-spanning b-barrel. The C-terminal domain (AIDA b-domain) inserts into the outer mem-brane as a b-barrel-like structure, and transports the N-terminallylinked passenger domain to the surface of the gram-negativeorganism (15). AIDA has advantages for recombinant surfacedisplay compared with other autotransporters, such as IgA1protease, due to its high efficiency for expression (16).

DNA vaccination remains a promising approach to stimulateCD8þ T-cell response (17, 18). How the DNA vaccine is deliveredto the host immune system is critical for induction of an effectiveantigen-specific immune response. DNA-encoding specific epi-topes can be incorporated as a live vaccine delivered by attenu-ated, nonreplicating Salmonella typhimurium (SL7207), whichtransport the DNA vaccine through DCs and macrophages(19–22). The attenuated bacteria are captured by antigen-pre-senting cells and die due to an internal mutation, liberatingmultiple copies of DNA, and activating CD8þ T-cell responses(23, 24).Meanwhile, antigen expressed onbacterial surface canbe

1Institute of Blood and Marrow Transplantation, Department of Hematology,Collaborative Innovation Center of Hematology, the First Affiliated Hospital ofSoochow University, Suzhou, P.R. China. 2Immunology Programme, LifeSciences Institute and Department of Microbiology and Immunology, NationalUniversity of Singapore, Singapore, Singapore. 3College of Basic Medicine andBiological Sciences, Medical Department, Soochow University, Suzhou, P.R.China. 4Animal Infectious Disease Laboratory, School of Veterinary Medicine,Yangzhou University, Yangzhou, P.R. China.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Y. Mei and L. Zhao contributed equally to this article.

CorrespondingAuthor:Haiyan Liu, National University of Singapore, 28MedicalDrive, Singapore 117456, Singapore. Phone: 65-6516-6661; Fax: 65-6778-2684;E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-16-0240-T

�2017 American Association for Cancer Research.

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presented by these APCs to stimulate CD4þ T cells (25, 26).Bacterial cell wall (LPS) and the bacterial DNA (nonmethylatedCpG motifs) act as adjuvants to enhance the antitumor immuneresponses (27, 28).

In the current study, we developed a tumor vaccine delivered bylive attenuated Salmonella that could activate both tumor-specificCD4þ T and CD8þ T-cell responses. The vaccine contained anAIDA-1 autotransporter withmurinemelanoma antigenMelan-Ainserted at its N-terminal domain (for class II presentation toCD4þ T cells) and a DNA vaccine element encoding two murinemelanoma class I epitopes (TRP-1: TWHRYHLL and TRP-2:SVYDFFVWL), for activation of CD8þ T cells. In tumor-bearingmice, intranasal administration of the vaccine could significantlyinhibit melanoma tumor growth. The Salmonella tumor vaccineenhanced both tumor-specific Th1 and CTL responses. It alsofacilitated the tumor infiltration of the T cells and promoted Th1cytokines production of both CD4þ and CD8þ T cells. Admin-istration of the vaccine significantly prevented lung metastasis ofthe tumor through similar mechanisms. In conclusion, our studyprovides a novel strategy for developing vaccines that can stim-ulate both Th1 and CTL responses by mucosal immunization,which may have general applicability to the development ofvaccines against tumors.

Materials and methodsAnimals

Specific-pathogen-free 6-week-old female C57BL/6 mice wereobtained from the Shanghai Laboratory Animal Center (Shang-hai, China). Mouse care and experimental procedures were per-formed under specific-pathogen-free conditions. All animal pro-tocols were approved by the Institutional Laboratory Animal Careand Use Committee at Soochow University.

Cell linesB16-F10 cells were obtained from the American Type Culture

Collection in 2011 and cultured in complete DMEM medium(Gibco) containing 10% FBS. It has not been authenticated, buttheMycoplasma testing was performed in July 2015 with negativeresults. B16-F10 cells were passaged every two days and the cellsused for experiments were not passaged more than three times.

Plasmid and vaccine constructionFor the empty vector construction, DNA element of CMV-

expressing cassette amplified by PCR using pVAX1 as templatewas inserted between the site of EcoRI and ClaI of the PMK90vector (Fig. 1A; Takara; ref. 29). The minigene vector was con-structed based on the empty vector. Briefly, the AIDA-1 fragmentwas replaced by Melan-A encoding fragment, the DNA elementencoding two melanoma CD8þ T-cell epitopes (TRP-1:TWHRYHLL and TRP-2: SVYDFFVWL) linkedwithDNA encodingAAY was subcloned to CMV-expressing cassette of the emptyvector. The vaccine vector was similar to the minigene vector withthe addition of AIDA-1 autotransporter. The empty, minigene,and vaccine vectors were transformed into Salmonella strainSL7207 (kindly provided by Dr. Bruce Stocker, Stanford Univer-sity School of Medicine; ref. 30), and the transformants arereferred to as vector, minigene, and vaccine, respectively.

Detection of Melan-A surface expressionRecombinant bacteria were grown on LB agar plates at 37�C

for 16 hours; then the bacteria were harvested and resuspended

in PBS with OD575 at 10.0. Suspension was incubated at 37�Cfor 10 minutes with or without trypsin (50 g/mL). Suspensionwas washed twice in PBS and the pellets were subsequentlyanalyzed using Melan-A monoclonal antibody (Abcam) byWestern blot.

Animal modelsIn the subcutaneous model, mice were subcutaneously inoc-

ulated with 5 � 104 B16-F10. Seven days later, mice were ran-domly divided into four groups: (i) medium group, mice wereintranasally inoculated with 20 mL NaHCO3; (ii) vector group,mice were intranasally inoculated with 1 � 108 CFU (20 mL)Salmonella typhimurium SL7207 carrying empty vector; (iii) mini-gene group, mice were intranasally inoculated with 1 � 108 CFU(20 mL) Salmonella typhimurium SL7207 carrying minigene vector;and (iv) vaccine group,micewere intranasally inoculatedwith1�108 CFU (20 mL) Salmonella typhimurium SL7207 carrying thevaccine plasmids. Tumors were measured every 2 days. Tumorvolumes were calculated using the formula V ¼ 1/2 (L � W2),where Lwas the length (longest dimension) andWwas the width(shortest dimension).

In themetastasis model, mice were intravenously injected with2 � 105 B16-F10 from the tail vein. Seven days later, mice weredivided into four groups and underwent the same treatmentdescribed in the subcutaneous model. After another 7 days, micewere sacrificed and lungs were removed, and the metastaticnodules were quantified.

In vitro T-cell proliferation assayDCs isolated from spleens were loaded with either Melan A

(Biovision) or B16-F10 cell lysate for about 24 hours. CD4þ andCD8þ T cells (25,000) were cocultured with 5,000 irradiatedMelanA-loaded DCs or B16-F10 cell lysate–loaded DCs in a96-well cell culture plate (Corning Costar), respectively. The cellswere cultured in RPMI 1640 medium supplemented with 10%fetal bovine serum and 20 IU/mL recombinant IL2 (Beijing FourRings Bio-Pharmaceutical). After 72 hours, cells were labeledwith3H-thymidine (Shanghai Institute of Physics, Chinese AcademyofSciences, Shanghai, China). Sixteen hours thereafter, incorpo-ration of 3H-thymidine was determined in quadruplicate, usinga liquid scintillation counter (PerkinElmer Instruments).

Cytotoxicity assayThe CytoTox 96 nonradioactive cytotoxicity assay (Promega)

was used to measure the cytotoxic activity of the splenocytes,according to the manufacturer's protocol. Briefly, target cells B16-F10 (6 � 103 cells/well) were plated on 96-well U-bottom plates(Corning Costar), and the splenocytes (effectors) were added to afinal volume of 100 mL at the ratios of 100:1, 50:1, and 25:1 (E:Tratio). The plates were then incubated for 6 hours in a humidified5% CO2 chamber at 37�C and centrifuged at 500 � g for 5minutes. Aliquots (50 mL) were transferred from each well tofresh 96-well flat-bottom plates, and an equal volume of recon-stituted substrate mix was added into each well. The plates werethen incubated in the dark at room temperature for�30minutes.Stop solution (50 mL) was added, and the absorbance wasmeasured at 492 nm. The percentage of killing at each E:T ratiowas calculated using the following formula: (A492nm[experimental] � A492nm [effector spontaneous] �A492nm[target spontaneous]) � 100/(A492nm [target maximum] �A492nm [target spontaneous]).

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Cell preparation from spleens, tumors, and bronchoalveolarlavage

Splenocytes were prepared by gently homogenizing the tissuesto release the cells. Debris were removed by filtering through a 70-mmnylonmesh. TILs were prepared by processing the tissues intosingle-cell suspensions, and lymphocytes were separated on a40%Percoll (GEHealthcare) gradient. Red blood cells were lysed.Cell counts were performed on a Coulter Z1 cell counter (Beck-man Coulter). For BAL preparation, lungs were lavaged via acannula inserted into the trachea and instilled with 2 mL PBS.

Flow cytometryMonoclonal antibodies (mAb) to murine proteins were pur-

chased fromBDBiosciences: FITC-conjugated anti-CD4 (RM4-5),FITC-conjugated anti-NK1.1 (PK136), FITC-conjugated anti-CD19 (1D3), FITC-conjugated anti-IL2 (JES6-5H4), PE-conjugat-ed anti-CD3e (145-2C11), PE-conjugated anti-IL4 (11B11), PE-conjugated anti-IFNg (XMG1.2), PerCP Cy5.5–conjugated anti-CD8a (53-6.7), PerCP Cy5.5–conjugated anti–Gr-1 (RB6-8C5),PE-Cy7–conjugated anti-CD11b (M1/70), PE-conjugated anti-

CD44 (IM7), FITC-conjugated anti-CD62L (MEL-14), PE-Cy7–conjugated anti-CD11c (HL3), or APC-conjugated anti-TNFa(MP6-XT22). Cells were incubated in FACS buffer (1 � PBS,1% BSA, and 0.1% NaN3) in the presence of purified anti-CD16/32 at saturation to block unspecific staining for 30minutesat 4�C. The flow cytometric results were analyzed with FACSCantoII (BD Biosciences) using CellQuest software.

In vivo CD4þ or CD8þ T-cell depletionTo deplete CD4þ or CD8þ T cells, mice received i.p. injections

of 1mgGK1.5 (rat anti-mouseCD4mAb, SungeneBiotech) or 53-6.7 (rat anti-mouse CD8 mAb, Sungene Biotech) 2 days beforeadministration of vaccines in the therapeutic model, and theantibody injections were repeated on day 5 after the first vacci-nation. The efficacy of cell depletion was confirmed by flowcytometric analysis of the spleens.

Immunohistochemistry and histopathologyFollowing euthanasia, tumor tissues were removed aseptically

and immediately fixed in 4% formalin at room temperature for

Figure 1.

Construction of the vaccine vectors. A, Schematic representation of the empty vector, minigene vector, and vaccine vector. B, Melan-A is expressed on theouter membrane. Surface expression of Melan-A was analyzed by trypsin treatment and Western blot analysis. C, Cytotoxicity assay using the splenocytesstimulated with the Salmonella tumor vaccine, minigene vaccine, vector control or medium. Splenocytes harvested from C57BL/6 mice were incubatedwith medium, recombinant Salmonella SL7207 harboring vaccine vector, minigene vector or the empty vector, and gentamycin was added to kill theintracellular bacteria. After washing with PBS, splenocytes were cultured for 5 days, and used as effector cells. Their killing capacity against B16-F10 cellswas measured using CytoTox 96 nonradioactive cytotoxicity assay kit. The assays were done in quadruplicates. The data shown are the representative ofat least three experiments. � , P < 0.05.

Tumor Vaccine Activating Both CD4þ and CD8þ T-cell Responses

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2 days. The fixed tissues were processed through graded concen-trations of ethanol and xylene and were then embedded inparaffin wax. Tissue sections of 4 to 5 mm were mounted onadhesive glass slides and were stained with H&E. Tumor sectionswere then deparaffinized and treated with 0.08% H2O2 for 30minutes to block endogenous peroxidase. Slides were incubatedwith rat anti-CD4 (GK1.5; Abcam) or rat anti-CD8 (2.43; Abcam)at 4�C overnight, followed by incubation with HRP-conjugatedrabbit anti-rat IgG (Abcam). Diaminobenzidine was used todevelop the staining reaction, and nuclear counterstaining wasperformed with hematoxylin (Solarbio). Slides were coded andexamined by a pathologist who was blinded for the experimentalhistory of the animals.

ELISAPathogen-free 6-week-old female C57BL/6 mice were intrana-

sally inoculated with either 20 mL NaHCO3 or 1 � 108 CFU (20mL) Salmonella typhimurium SL7207 carrying the vaccine plasmids.Two and 7 days later, mice were sacrificed and BAL was collectedfrom mice. IL6, TNFa, and IFNg concentrations were measuredusing mouse IL6, TNFa, and IFNg Quantikine ELISA Kit (R&DSystems).

Statistical analysisAll data were analyzed with one-way ANOVA and were

expressed as means � SEM; data were analyzed using GraphPadPrism 5 software for Windows (GraphPad Software), and differ-ences were considered statistically significant when P < 0.05. Thesignificance levels aremarked �,P<0.05; ��,P<0.01; �� P<0.001.

ResultsConstruction of the Salmonella tumor vaccine

The expression vector was designed based on the pMK90 vectorby insertion of a CMV-expressing cassette. The empty vectorcontains two elements. The AIDA-1 autotransporter elementexpressed the passenger protein, for class II presentation, on thegram-negative bacterial surface under control of the constitutiveAIDA-1 promoter. The CMV-expressing cassette could expressantigens in eukaryotic cells (Fig. 1A). The vaccine vector wasdesigned based on the empty vector. TheMelan-A sequences wereinserted to theN-terminal of AIDA-1 element, and genes encodingtwoCD8þ T epitopes (Trp-1and Trp-2) ofmurinemelanomawerecloned into the CMV-expressing cassette (Fig. 1A). The minigenevector was similar to the vaccine except it lacked the AIDA-1autotransporter. The vaccine vector,minigene vector, or the emptyvector were then transformed into attenuated Salmonella strainSL7207.

As surface-exposed protein can be cleaved off during exposureto trypsin, whereas cytoplasmic or periplasmic protein are notaffected, the surface expression of Melan-A was confirmed bytrypsin treatment of intact bacteria and Western blotting of thebacteria (Fig. 1B). Melan-A expression could be detected on thesurface of the vaccine bacteria before trypsin treatment while noMelan-A expression was seen after the treatment (Fig. 1B).

To examine whether the vaccine could enhance the antitumoractivity of the lymphocytes in vitro, the cytotoxicity of splenocytesstimulated with medium or Salmonella transduced with emptyvector, minigene, or vaccine vector against B16-F10 cells wasmeasured (Fig. 1C). Splenocytes stimulated with medium orcontrol Salmonella showed similarly low cytotoxicity. Stimulation

with the Salmonella expressing minigene slightly increased cyto-toxicity, but with no statistical significance. Notably, splenocytesstimulated with Salmonella vaccine exhibited significantly highercytotoxicity against B16-F10 cells than the medium and vectorgroups. These results indicated that the Salmonella tumor vaccinewas successfully constructed and it could enhance the antitumorcytotoxicity of the splenocytes in vitro.

Suppression of primary tumor growth by Salmonellatumor vaccine

Toexamine the antitumor effect of the Salmonella tumor vaccinein vivo, C57BL/6mice were injected subcutaneously with B16-F10cells (5 � 104). On day 8, tumor-bearing mice were intranasallyadministrated with 1 � 108 recombinant SL7207 carrying emptyvectors (vector group), recombinant SL7207 carrying minigenevectors (minigene group), recombinant SL7207 carrying vaccinevectors (vaccine group), or medium alone. Tumor volumes weremonitored every other day. Tumor growth was similar betweenthe vector group and the medium group (Fig. 2A). Minigenetreatment slightly reduced the tumor growth, but was not statis-tically significant. Tumor growth was significantly inhibited inmice that received Salmonella tumor vaccine, compared with theother three groups. To demonstrate the safety of the Salmonellatumor vaccine, we monitored the weight of the tumor-bearingmice before and after vaccine inoculation. Mice of all groupsshowed similar body weights and similar health conditions (Fig.2B), demonstrating that the attenuated Salmonella tumor vaccinedid not cause weight loss or any other major side effects. We alsodetected significant increases of IL6, TNFa, and IFNg in lungtissues using ELISA on day 2 after vaccination compared with themedium control (Fig. 2C). However, these inflammatory cyto-kines were back to basal concentrations on day 7, suggesting atransient inflammatory reaction induced by the vaccination.

In order to elucidate the possible mechanisms of antitumoreffects induced by Salmonella tumor vaccine, we first analyzed thetotal numbers of tumor-infiltrating lymphocytes (TIL). TILs pertumor weight were significantly increased in the vaccine groupcomparedwith themedium, vector, orminigene groups (Fig. 2D).However, on day 16 after tumor inoculation, the percentages andtotal numbers of T cells, B cells, NK cells, NKT cells, DCs, macro-phages, and MDSCs were similar in spleens from the four treat-ment groups (Fig. 2E). The percentages and total numbers ofCD4þ T and CD8þ T cells were not significantly different either.The percentages and numbers of the tumor-infiltrating immunesubsets were also similar among four treatment groups (Fig. 2E).Therefore, although TILs were slightly increased in the vaccinegroup, no cell subset was significantly increased in percentage.

Tumor-specific CD4þ and CD8þ T cells are critical for theantitumor effects

To further evaluate the function of the T cells after Salmonellatumor vaccine treatment, intracellular staining was performedwith splenocytes and TILs to examine the tumor-specific T-cellresponse (Fig. 3A–C; Supplementary Fig. S1). Both CD4þ andCD8þ T cells from the vaccine group had significantly increasedTNFa and IFNg production compared with the control groups,whereas only CD4þ T cells had increased IL2 expression in thespleen (Fig. 3A and B). Similarly, the percentages of both TNFa-producing CD4þ T and CD8þ T cells in TILs were significantlyhigher in the mice of the vaccine group than in those of the other

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Figure 2.

Therapeutic effects of the Salmonella tumor vaccine in subcutaneous melanoma model. A, Tumor growth of melanoma-bearing mice. C57BL/6 mice wereinoculated s.c. at the right flank with 5 � 104 B16-F10 cells. Seven days later, mice were intranasally inoculated with 20 mL NaHCO3, 1 � 108 CFU (20 mL)Salmonella SL7207 carrying the empty vector, minigene vector, or 1 � 108 CFU (20 mL) Salmonella SL7207 carrying the vaccine vector. B, The weightchanges of the tumor-bearing mice were monitored every day. C, IL6, TNFa, and IFNg were detected by ELISA in the lung tissues of the mice treated withmedium or Salmonella SL7207 carrying the vaccine vector. D, Mice were sacrificed on day 14 post tumor inoculation, lymphocyte numbers per tumorweight were analyzed. E, Frequencies and total numbers of T, B, CD4þ T, CD8þ T, DCs, macrophages, MDSCs, NK, and NKT cells in the spleen and TILwere analyzed. The experiments were performed with six mice per group. The data shown are the representative of three experiments. � , P < 0.05; �� , P < 0.01;�� , P < 0.001.






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groups, whereas only CD4þ T cells from the vaccine group hadincreased IL2 production (Fig. 3C). These data suggested thatSalmonella tumor vaccine promoted both tumor-specific CD4þ

andCD8þ T-cell responses in terms of Th1 cytokine and IL2 (fromCD4þ T cells) productions.

In order to confirm the enhancement of tumor-specific CD4þ

and CD8þ T-cell responses, T-cell proliferation assays were per-formed with the stimulation of irradiated DCs pulsed withMelanA (for CD4þ T cells) or B16-F10 tumor lysates (for CD8þ T cells).The proliferation of CD4þ T cells specific to Melan A was signif-icantly increased in the vaccine group than the vector, minigene,or medium group (Fig. 3D). On the other hand, CD8þ T-cellproliferation showed no difference among four groups (Fig. 3D).The percentages of Trp-2-specific CD8þ T cells in TILs wereexamined by tetramer staining. The percentages of tumor antigenTrp-2–specific CD8þ T cells in TILs were significantly increased invaccine group compared with the vector, minigene, or mediumgroup (Fig. 3E). To measure tumor-specific cytotoxicity, totalsplenocytes from eachmouse were harvested and stimulated withB16-F10 cell lysates in the presence of IL2 (Fig. 3F). After 5 days ofincubation, effector cellswere coculturedwith B16-F10 target cellsat various E/T ratios. Splenocytes from mice that received Salmo-nella tumor vaccine showed higher killing capacity against B16-F10 cells compared with those frommice of the vector, minigene,or medium group (Fig. 3F). All these results demonstrated thatSalmonella tumor vaccine promoted both tumor-specific CD4þ

and CD8þ T-cell responses.To determine whether CD4þ and CD8þ T cells were critical for

the antitumor activity induced by the Salmonella tumor vaccine,weperformed in vivodepletionofCD4þ and/orCD8þT cells usingspecific mAbs (Fig. 3G). Depletion of CD4þ or CD8þ T cellssignificantly reduced the therapeutic effects of the vaccine. Thetherapeutic effects were further inhibited by depletion of bothCD4þ and CD8þ T cells. These results suggested that the antitu-mor response induced by Salmonella tumor vaccine was depen-dent on both CD4þ and CD8þ T cells.

Salmonella tumor vaccine prevents melanoma lung metastasisIn order to assess whether Salmonella tumor vaccine could also

protect mice from tumor metastasis, we established a murinemelanoma lung-metastasis model. Seven days after B16-F10 cellinoculation, mice were treated with Salmonella tumor vaccine,minigene vaccine, vector control, or medium intranasally (Fig.4A).Micewere sacrificed and lungswere removed7days later, andthe visible metastases were counted. Vaccine treatment dramat-ically reduced the number of lung metastases compared with thevector, minigene, and medium treatment, and minigene vaccinetreatment also significantly reduced tumor metastasis (Fig. 4A).The body weights of the mice were also monitored through the

course of experiments to demonstrate the safety of the Salmonellatumor vaccine (Fig. 4B).

TILs were phenotypically analyzed by flow cytometry on day 14after tumor inoculation (Fig. 4C). Increased tumor infiltrations ofCD8þ T cells were observed in mice immunized with Salmonellatumor vaccine compared with the vector, minigene, or mediumgroup. Total tumor-infiltrating T cells were comparably higher inboth tumor vaccine andminigene groups than those in the vectoror medium group. These results were also confirmed by H&Estaining and immunohistochemistry staining (Fig. 4D). CD8þ T-cell infiltration was greatest in the vaccine group compared withthe other two groups.

CD4þ and CD8þ T cells activated by Salmonella vaccine in lungmetastasis model

To further evaluate T-cell functions after the Salmonella tumorvaccine treatment, intracellular staining was performed afterstimulation with B16-F10 cell lysates to evaluate the tumor-specific T-cell response (Fig. 5; Supplementary Fig. S2). Thepercentages of TNFa- and IFNg-expressing CD4þ T cells in thespleens of the mice treated with Salmonella tumor vaccine weresignificantly higher than those of the mice treated with Salmonellaexpressing minigene, vector, or medium (Fig. 5A). The percen-tages of TNFa-, IFNg-, and IL2-expressing CD8þ T cells, as well asIL2-expressing CD4þ T cells, in the spleens were significantlyincreased in the vaccine group compared with the vector ormedium group. On the other hand, the percentages of TNFa-expressing CD4þ and CD8þ T cells in the TILs were significantlyhigher in the Salmonella tumor vaccine group than in the vector,minigene, or medium group, although there was no increase inIL2 production (Fig. 5B). TNFa and IL2 production by bothCD4þ

and CD8þ T cells in the BAL was significantly increased in thevaccine group, although IFNg production was similar among alltreatment groups (Fig. 5C). These results suggest that both tumor-specific CD4þ and CD8þ T cells were better activated and pro-ducing more Th1 cytokines and IL2 in the vaccine group.

In order to confirm the finding of T-cell activation by cytokineproduction, the proliferation of tumor-infiltrating CD4þ andCD8þ T cells upon Melan A or tumor lysate stimulation wereexamined by the 3H-thymidine incorporation assay (Fig. 6A). Theproliferations of both tumor-infiltrating CD4þ and CD8þ T cellswere significantly increased in Salmonella tumor vaccine groupthan in the vector or medium group. CD4þ T-cell proliferationfrom the vaccine group was also significantly higher than thosefrom the minigene group. We also detected the percentages oftumor antigen Trp-2-specific CD8þ T cells in TILs. An increasedpercentage of Trp-2-specific CD8þ T cells was observed in micethat received the vaccine treatment compared with the vector,minigene, or medium group (Fig. 6B). The killing capacity of the

Figure 3.Antitumor response induced by the Salmonella tumor vaccine was CD4þ and CD8þ T-cell dependent. Intracellular staining of TNFa, IFNg , and IL2 by gatingon CD4þ (A) or CD8þ (B) T cells from splenocytes stimulated with B16-F10 lysates. C, Intracellular staining of TNFa and IL2 by gating on CD4þ or CD8þ Tcells from TILs stimulated with B16-F10 lysates. D, Proliferations of CD4þ T and CD8þ T cells were measured by 3H-thymidine incorporation assay. E,Tetramer staining of Trp-2–specific CD8þ T cells in TILs. F, Cytotoxicity assay of splenocytes from the tumor-bearing mice. Splenocytes from treated micewere isolated on day 14 post tumor injection and then incubated with lysates of B16-F10 cells. After 5 days of incubation, the effector cells were coculturedwith B16-F10 at various E:T ratios using CytoTox 96 nonradioactive cytotoxicity assay. G, Tumor growth of vaccine-treated mice with depletion of CD4þ or CD8þ Tcells. In the depletion group, mice were i.p. injected with 1 mg GK1.5 (rat anti-mouse CD4 mAb) or 53-6.7 (rat anti-mouse CD8 mAb) 2 days before the firstadministration of the vaccine. Mice were sacrificed on day 17 post tumor inoculation and tumor volumes were measured. The experiments were performedwith six to eight mice per group. The assays were done in quadruplicates. The data shown are the representative of three experiments. � , P < 0.05; �� , P < 0.01;�� , P < 0.001.






Figure 4.

Therapeutic effects of Salmonella tumor vaccine in lung metastasis model. A, The numbers of metastatic nodules in the lungs of the tumor-bearing mice.Mice were i.v. injected with 2 � 105 B16-F10 from tail vein. 7 days later, mice were divided into 4 groups, and immunized the same dose of Salmonellatumor vaccine, minigene vaccine, vector control, or the medium. Seven days later, the lungs were removed from these mice, and the metastatic noduleswere quantified. B, The weight changes of the tumor-bearing mice were monitored every day. C, Mice were sacrificed on day 14 post tumor inoculation,frequencies of T, CD4þ T, and CD8þ T cells in the lungs were analyzed. D, H&E and immunochemistry staining for CD4þ and CD8þ T cells of tumortissues from melanoma-bearing mice on day 14 (�200 magnification). The data shown are the representative of at least three experiments. � , P < 0.05; �� , P < 0.01;�� , P < 0.001.

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Figure 5.

Salmonella tumor vaccine promoted tumor-specific Th1 cytokine and IL2 production of CD4þ and CD8þ T cells in the lung metastasis model. A, Intracellularstaining of TNFa, IFNg , and IL2 of CD4þ and CD8þ T cells from splenocytes in the lung metastasis model stimulated with B16-F10 lysates. B, Intracellularstaining of TNFa, IFNg , and IL2 of CD4þ and CD8þ T cells from TILs in the lung metastasis model stimulated with B16-F10 lysates. C, Intracellular staining of TNFa,IFNg , and IL2 of CD4þ and CD8þ T cells from BAL in the lung metastasis model stimulated with B16-F10 lysates. The experiments were performed with six toeight mice per group. The assays were done in quadruplicates. The data shown are the representative of three experiments. � , P < 0.05; ��, P < 0.01; �� , P < 0.001.






splenocytes from the vaccine group against B16-F10 tumor cellswas also significantly higher than those from the vector,minigene,or medium group at various E/T ratios (Fig. 6C). These resultsdemonstrated that the Salmonella tumor vaccine enhanced bothtumor-specific CD4þ andCD8þ T-cell responses in themelanomalung metastasis model.

DiscussionStimulation of both tumor-specific Th1 and CTL responses has

been one of the challenges for effective tumor vaccines. In thecurrent study, a vaccine vector containing AIDA-1 autotransporterand DNA vaccine elements was designed, and the murine mel-anoma vaccinewas generatedwith this vector anddelivered by theattenuated Salmonella strain SL7207. Our results demonstratedthat this vaccine strategy activated both tumor-specific Th1-polar-izedCD4þT cell andCTL responses againstmelanoma in vivo andpromoted antitumor immunity.

Recombinant attenuated Salmonella strains are promising car-riers for oral or intranasal delivery of antigens. Salmonellamainlysurvives in antigen-presenting cells, rendering surface antigensmore accessible to the cell for processing and presentation.

Surface-located antigens on Salmonella induce more efficientCD4þ T response than those expressed in cytoplasma (26). Forexample, oral administration of Salmonellawith surface display ofovalbumin epitopes couldpromote the activationofCD4þT cells.In H. pylori infection model, surface display of UreA epitopeincreased the immunogenicity and mediated a significant reduc-tion in the level of H. pylori in immunized mice, whereas con-ventional cytoplasmic expression of UreA in Salmonella had noeffect (11). Surface-exposed foreign epitopes are often presentedasmultimers and could induce better immune responses than thesame epitopes presented as monomers in a Salmonella vaccine(31). Therefore, antigen expressed on Salmonella surface usingsurface display system can induce better CD4þ T response. Auto-transporter is an efficient surface display system and many func-tional B-cell epitopes and T-cell epitopes have been successfullyexpressed using autotransporter (32–35). Among the differentautotransporters, AIDA-1may be themost efficient, as it has beenshown to express many heterologous proteins on gram negativebacterial outer membrane, including b-lactamase (12), Vibriocholera ctxB protein (36), and bovine adrenodoxin (37). Weharnessed the function of AIDA-1 system to deliver a tumorantigen (Melan-A) by the live attenuated vaccine strain in order

Figure 6.

Salmonella tumor vaccine promoted the function of both tumor-specific CD4þ T cells and CTLs in the lung metastasis model. A, Proliferations of CD4þ Tand CD8þ T cells from the spleens of tumor-bearing mice stimulated with B16-F10 lysates were measured by 3H-thymidine incorporation assay. B, Tetramerstaining of Trp-2–specific CD8þ T cells in TILs. C, Cytotoxicity assay of splenocytes from the tumor-bearing mice against B16-F10 tumor cells. Splenocytesfrom the tumor-bearing mice were isolated on day 14 post tumor injection and then incubated with lysates of B16-F10 cells. After 5 days of incubation, theeffector cells were cocultured with B16-F10 at various E:T ratios using CytoTox 96 nonradioactive cytotoxicity assay. The experiments were performedwith six to eightmice per group. The assayswere done in quadruplicates. The data shown are the representative of at least three experiments. � , P <0.05; �� , P <0.01;�� , P < 0.001.


Mei et al.




to stimulate CD4þ T-cell response. Indeed, the tumor antigen wasefficiently expressed in the tumor vaccine and a potent tumor-specific CD4þ T-cell response was induced. Our results of usingminigene vectorwithout the AIDA-1 system further demonstratedthe importance of surface displaying antigens for CD4þ T-cellactivation.

Different subpopulations of CD4þ T cells have different func-tions during host antitumor immune responses. Th1 cells exhibitantitumor effects by optimizing CTLs function through multipleinteractions during the induction and effector phases of antitu-mor immune responses as well as direct cytokine productions (9).On the other hand, Th2 and Treg cells exert inhibitory effects onthe antitumor immune response and promote tumor growth(38). The role of Th17 cells in tumor development is still con-troversial (39). In the vaccine group, the increased CD4þ T-cellproliferation was accompanied by significant higher levels ofTNFaproduction, indicating the Salmonella tumor vaccinepotent-ly promoted Th1 responses. The antigen-specific IL4 or IL17producing CD4þ T cells were too few to be detected, suggestingthat Th2 or Th17 responses were not induced by the vaccine. Thepercentage of Tregswas not significantly different among the threetreatment groups. These results suggested that CD4þ T-cellresponse induced by the AIDA-1 system was skewed to the Th1response, which was in accordance with the results obtained withattenuated Salmonella expressing Yersinia enterocolitica MHC-IIepitope (HSP6074-86) by AIDA-1 (34).

DNA-based vaccines have emerged as an attractive approach toactivate CD8þ T cells (40). However, the immunogenicity of theplasmid DNA needs to be improved. Among major contributorsto enhanced efficiency are vector design and optimization ofdelivery methods (41). Using oral or nasal delivery system withattenuated Salmonella is a promising method to enhance theimmunogenicity of the DNA vaccine. Immunization with atten-uated Salmonella harboring plasmid DNA vaccines proved to beeffective in DNA delivery and subsequent induction of immunityagainst the encoded antigens. A previous study found that DNAvaccines delivered by attenuated Salmonella SL7207 induced astronger cellular antitumor immune response than gene gunapproaches, or injection of lentivirally transduced bone mar-row-derived DCs (19). Our study revealed that the tumor DNAvaccine delivered by Salmonella promoted the proliferation ofCD8þ T cells in vivo. It also induced a significantly higher per-centages of TNFa- and IFNg-producing CD8þ T cells and antigen-specific CD8þ T cells in TILs, indicating an effective CD8þ T-cellstimulation by the Salmonella tumor vaccine.

Insertion of heterologous genes to a live vaccine strain is ametabolic burden, and often leads to suboptimal antigen deliveryand rapid loss of the expression vector, which decreases theefficiency of the vaccine (42). Attenuated Salmonella vaccinedisplayingheterologous antigenbyMisL autotransporter required

multiple immunizations due to the instability of the vaccine (35).In our study, the insertion of foreign genes to the vaccine vectorseemed to be well tolerated by the attenuated Salmonella, as theantitumor immune responses were evoked by administration ofonly one dose of vaccine.

In order to stimulate both CD4þ and CD8þ T-cell responses,multiple immunizations with different types of vaccines, such aspriming with DNA vaccine followed by boosting with proteins orpeptides, are often needed (43). Long peptides encompassingboth Th epitopes and CTL epitopes have been designed tostimulate both CD4þ and CD8þ T cell responses (44). However,these methods require appropriate adjuvants, multiple immuni-zations, and intradermal or intramuscular injections, and aretime-consuming, costly, and potentially hazardous because ofthe parenteral injections. In the current study, we described aneffective cancer vaccine delivered by attenuated Salmonella thatcould activate both Th1 and CTL responses. This strategy could bewidely applied in the development of oral or nasal vaccinesagainst tumors.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: L. Zhao, H. LiuDevelopment of methodology: Y. Mei, L. Zhao, Y. Liu, H. LiuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Mei, L. Zhao, Y. Liu, H. Gong, Y. Song, L. Lei,Y. Zhu, Z. Jin, S. Ma, Q. SunAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Mei, L. Zhao, Z. Jin, H. LiuWriting, review, and/or revision of the manuscript: Y. Mei, L. Zhao, H. LiuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. Zhao, B. HuStudy supervision: H. Liu

Grant SupportThis work has been supported by grants from the National Natural Science

Foundation of China (81471586, 31500746), the project funding from Suzhoucity (SWG0904), the Priority Academic Program Development of JiangsuHigher Education Institutions, the Natural Science Foundation of the JiangsuHigher Education Institutions of China (15KJB320015), the Open ProjectProgram of Jiangsu Key Laboratory of Zoonosis (R1601), the Training Programsof Innovation and Entrepreneurship for Undergraduates of SoochowUniversity(201510285105X), the Industrial technology innovation project of Suzhou(SYS201677), and the start-up grant from the National University of Singapore.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 16, 2016; revisedMarch 1, 2017; accepted April 21, 2017;published OnlineFirst May 3, 2017.

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2017;5:503-514. Published OnlineFirst May 3, 2017.Cancer Immunol Res Yu Mei, Lixiang Zhao, Yonghao Liu, et al.

T-cell Responses+ and CD8+Activates Tumor-Specific CD4SalmonellaCombining DNA Vaccine and AIDA-1 in Attenuated

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