Fusion of the dendritic cell-targeting chemokine MIP3α to melanoma antigen Gp100 in a therapeutic DNA vaccine significantly enhances immunogenicity and survival in a mouse melanoma model
© The Author(s). 2016
Received: 23 May 2016
Accepted: 7 November 2016
Published: 20 December 2016
Although therapeutic cancer vaccines have been mostly disappointing in the clinic, the advent of novel immunotherapies and the future promise of neoantigen-based therapies have created the need for new vaccine modalities that can easily adapt to current and future developments in cancer immunotherapy. One such novel platform is a DNA vaccine fusing the chemokine Macrophage Inflammatory Protein-3α (MIP-3α) to an antigen, here melanoma antigen gp100. Previous published work has indicated that MIP-3α targets nascent peptides to immature dendritic cells, leading to processing by class I and II MHC pathways. This platform has shown enhanced efficacy in prophylactic melanoma and therapeutic lymphoma model systems.
The B16F10 melanoma syngeneic mouse model system was utilized, with a standard therapeutic protocol: challenge with lethal dose of B16F10 cells (5 × 104) on day 0 and then vaccinate by intramuscular electroporation with 50 μg plasmid on days three, 10, and 17. Efficacy was assessed by analysis of tumor burden, tumor growth, and mouse survival, using the statistical tests ANOVA, mixed effects regression, and log-rank, respectively. Immunogenicity was assessed by ELISA and flow cytometric methods, including intracellular cytokine staining to assess vaccine-specific T-cell responses, all tested by ANOVA.
We demonstrate that the addition of MIP3α to gp100 significantly enhances systemic anti-gp100 immunological parameters. Further, chemokine-fusion vaccine therapy significantly reduces tumor burden, slows tumor growth, and enhances mouse overall survival compared to antigen-only, irrelevant-antigen, and mock vaccines, with efficacy mediated by both CD4+ and CD8+ effector T cells. Antigen-only, irrelevant-antigen, and chemokine-fusion vaccines elicit significantly higher and similar CD4+ and CD8+ tumor-infiltrating lymphocyte (TIL) levels compared to mock vaccine. However, vaccine-specific CD8+ TILs are significantly higher in the chemokine-fusion vaccine group, indicating that the critical step induced by the fusion vaccine construct is the enhancement of vaccine-specific T-cell effectors.
The current study shows that fusion of MIP3α to melanoma antigen gp100 enhances the immunogenicity and efficacy of a DNA vaccine in a therapeutic B16F10 mouse melanoma model. This study analyzes an adaptable and easily produced MIP3α-antigen modular vaccine platform that could lend itself to a variety of functionalities, including combination treatments and neoantigen vaccination in the pursuit of personalized cancer therapy.
KeywordsDNA Vaccine MIP3α, MIP3alpha, or CCL20 B16 Melanoma Gp100 Therapeutic cancer vaccine Chemokine-antigen fusion In vivo electroporation
The recent therapeutic success of immunotherapies  and the identification of cancer neoantigens as potential therapeutic targets [2, 3] have generated renewed interest in the field of cancer vaccines. Although only one therapeutic cancer vaccine is currently FDA-approved (Sipuleucel-T ), hypothesized synergies between current and future immunotherapies  have increased the need for new vaccine platforms that can best address the new immunotherapeutic opportunities.
DNA vaccines offer many advantages as cancer therapies. They generate effector immunity from all three arms of the adaptive immune response, particularly including CD8+ T-cells . They avoid the inclusion of extraneous and possible deleterious antigens that may be components of bacterial or viral-based vaccines . They stimulate innate immunity and avoid issues of safety and practicality associated with various vectors . They can also be readily adapted to novel or mutating antigenic targets, are stable at room temperature, and can be constructed quickly . Clinical trials with a variety of antigens have demonstrated safety and immunogenicity of clinical DNA vaccines [7, 8]. However, initial trials for therapeutic DNA cancer vaccines have all shown limited effectiveness . More recent advances in DNA vaccination modalities have rekindled interest in their potential efficacy for cancer therapy [10, 11]. Of note, DNA vaccines have shown efficacy in animals, with three licensed for veterinary use [12–14].
One of the primary hurdles for DNA vaccines has been their limited potency in the clinical setting . Novel approaches to in vivo DNA delivery are being developed to address this issue. In vivo electroporation has been shown in animal models to enhance the breadth and potency of elicited immune responses [15–18]. Mechanistic studies have shown electroporation increases DNA uptake, stimulates local inflammation at the vaccination site, and enhances amount of vaccine antigen produced in situ [19–21]. In vivo electroporation is currently being utilized in the veterinary clinic as a mode of introducing a hormone into pregnant sows  and is currently undergoing clinical trials [23, 24].
Additionally, investigators have been taking advantage of the inherent flexibility of DNA to add immunomodulators to the vaccine construct in order to enhance the efficiency of initiating a specific immune response. Many studies have focused on increasing productive contact of nascent vaccine antigens to antigen presenting cells (APCs), especially dendritic cells (DCs). One approach is to fuse antigens to cytokines such as GM-CSF that can stimulate the development, proliferation, and maturation of DCs and monocytes [25–27] or to chemokines like CCL5 , CCL19 , MIP3α (also known as CCL20) [30–35], or other molecules [36–40] that can recruit and/or target nascent peptides to APCs. MIP3α fusion vaccines have been shown to direct antigen to immature DCs via CCR6 and mediate antigen uptake in a fusion dependent manner , after which, antigens are cross presented by both MHC class I and II, activating significant responses from both CD4+ and CD8+ T cells [31–33].
In the current studies, a DNA vaccine administered by intramuscular electroporation with a construct fusing MIP3α to the melanoma tumor-associated antigen gp100 has been analyzed in a therapeutic vaccination protocol utilizing the B16F10 melanoma mouse model system. MIP3α-antigen fusion DNA vaccine constructs have shown efficacy in a prophylactic melanoma model against gp100 , a therapeutic lymphoma model against oncofetal antigen (OFA) , and a prophylactic malaria model against circumsporozoite protein (CSP) . Here we compare therapeutic MIP3α-gp100 vaccination to a construct with a mutated MIP3α sequence that abrogates its function, effectively providing a gp100 antigen-only vaccine, and to a construct fusing the chemokine to an antigen irrelevant to this system, CSP. These experiments show that inclusion of functional MIP3α in the vaccine construct used in the therapeutic protocol enhances immunogenicity, slows tumor growth, and significantly extends survival compared to antigen-only and irrelevant-antigen vaccinations.
Animals and tumor model
Five to six week old female C57BL/6 (H-2b) mice were purchased from Charles River Laboratories (Wilmington, MA) and maintained in a pathogen-free micro-isolation facility in accordance with the National Institutes of Health guidelines for the humane use of laboratory animals. All experimental procedures involving mice were approved by the IACUC of the Johns Hopkins University (Protocol number MO13H219 and MO16H85). B16F10 mouse melanoma cells were a generous gift from Dr. Arya Biragyn (NIH, Baltimore, MD). Six to eight week old mice were challenged in the left flank subcutaneously with a lethal dose (5 × 104 cells) of B16F10 melanoma. Tumor size was recorded as square mm, representing tumor length × width (opposing axes) measured by calipers every 1–3 days. Mice were kept in the study until one of the following occurred: mouse death, tumor size eclipsing 20 mm in any direction, or extensive tumor necrosis resulting in excessive bleeding.
Plasmids and vaccination
Vaccine consisted of purified plasmid DNA in endotoxin-free PBS. The plasmid encoded either MIP3α-gp100, MIP3α-CSP as described , or dMIP3α-gp100 fusion sequence as described . dMIP3α-gp100 vaccine DNA is identical except for a point mutation in the chemokine changing a structurally necessary cysteine to serine (C6S), which abrogates chemokine functionality . Vaccination plasmid was extracted from E. coli using Qiagen® (Germantown, Md) EndoFree® Plasmid Maxi and Giga Kits. Vaccine DNA purity, quality, and quantity were verified by gel electrophoresis, restriction enzyme analysis, Nanodrop® spectrophotometry, and full insert sequencing. Mock vaccinations comprised of endotoxin-free PBS only. DNA injections were administered into the hind leg tibialis muscle. Immediately following injection, the muscle was pulsed using an ECM 830 Electro Square Porator™ with 2-Needle Array™ Electrode (BTX Harvard Apparatus®; Holliston, MA) under the following parameters: 106 V; 20 ms pulse length; 200 ms pulse interval; 8 total pulses. Vaccinations of 50ug/dose were delivered at days noted in figure legends. Prophylactic efficacy of the vaccine was confirmed, replicating previously reported results in which DNA was delivered by gene gun  [Additional file 1]. Vaccine DNA was also confirmed to express specific protein after transfection into Hek-293 T cells [Additional file 2], as detected by Western blot analysis using antibodies targeting the myc tag present at the 3′ end of the construct. As described by others, vaccination for the therapeutic model began on day three [41, 42].
In cell ELISA
Humoral immune responses to the vaccine were tested by an In-Cell ELISA assay to detect overall response to native B16F10 proteins, including gp100. The studies utilized the standard protocol for In-Cell ELISA from Abcam® (Cambridge, UK). In brief, wells of tissue-culture treated 96-well plates were seeded with 5 × 104 B16F10 cells and were allowed to adhere for 3–4 h at 37 °C. Adherence was verified by microscopy before proceeding. Cells were fixed, incubated with serum or primary control antibody (rabbit anti-gp100 ab137078 [Abcam, Inc.; Cambridge, UK]) at varying dilutions overnight at 4 °C, blocked with 2% BSA, and then incubated with peroxidase-conjugated goat anti-mouse IgG (serum) or goat anti-rabbit IgG(control) (Jackson ImmunoResearch Laboratories, West Grove, PA) at a dilution of 1:5000. Wells were developed for 1 h using ABTS® ELISA HRP Substrate (KPL, Gaithersburg, MD). The data were collected using the Synergy™ HT (BioTek Instruments, Inc. Winooski, VT).
Extraction of splenocytes and TILs
Spleen and tumor cell suspensions were prepared by grinding sterile excised tissue between the frosted ends of microscope slides and then passing the tissue through a sterile 60 μM mesh. Splenocytes were processed by lysing red blood cells and washing with sterile PBS. Tumor lysate was washed with sterile PBS, and tumor-infiltrating lymphocyte (TIL) fraction was enriched by Lympholyte®-M Cell Separation Media (Cedarlane®, Burlington, NC) according to the manufacturer’s protocol. Prior to use all cells were counted by a Z1™ Coulter Counter® (Beckman Coulter, Inc.; Brea, CA) and/or a hemocytometer with Gibco™ Trypan Blue solution 0.4% (Life Technologies, Carlsbad, CA).
Intracellular cytokine staining and flow cytometry
Enriched splenocytes or TILs were seeded onto Falcon® Multiwell 24-well tissue culture treated plates (Corning, Inc.; Corning, NY) at 1 × 106 cells per well (or all cells if total is less) and stimulated for 3–4 h at 37 °C with known immunodominant gp10025-33 (KVPRNQDWL) peptide or control HA (YPYDVPDYA) peptide (JHU School of Medicine Synthesis & Sequencing Facility; Baltimore, MD) combined with Protein Transport Inhibitor Cocktail and costimulatory anti-CD28 and anti-CD49d agonizing antibodies (eBioscience, Inc. San Diego, Ca). Cells were collected, washed, fixed, permeabilized, and stained using standard laboratory protocols for intracellular staining. Fixation and permeabilization buffers from Mouse Regulatory T Cell Staining Kit #2 (eBioscience, Inc. San Diego, Ca) were used. Stains utilized were the following anti-mouse mAbs: PercPCy5.5 conjugated anti-CD3, APC-conjugated anti-IFNγ, FITC-conjugated anti-CD8, and PE-conjugated anti-CD4 (eBioscience, Inc. San Diego, CA). Utilized FACSCalibur™ and LSRII™ Flow Cytometers (BD Biosciences, San Jose, CA). Flow Data analyzed by FlowJo Software (FlowJo, LLC Ashland, OR).
To deplete the CD4+, CD8+, or both T cell subsets, immunized mice were injected i.p. with anti-CD4 (GK1.5), anti-CD8 (2.43), or both mAbs, which were generous gifts from Dr. Fidel Zavala (JHSPH, Baltimore, MD). Negative control vaccinated mice received isotype Rat IgG2b antibody against KLH (LTF-2) purchased from BioXCell (West Lebanon, NH). 100 μg of antibody was given to each mouse i.p. on days -1, 0, and 7 from tumor challenge. Depletion efficacy was tested on days 0 and 8 or 10 by two-color flow cytometry analysis of peripheral blood lymphocytes using a FACSCalibur™ cytometer (BD Biosciences, San Jose, Ca) with FITC conjugated anti-mouse CD4 and APC-conjugated anti-mouse CD8 mAbs (eBioscience, Inc. San Diego, Ca).
Statistics and availability of data
Tumor size, immunologic, and flow cytometric analyses were statistically tested by one-way ANOVA with Bonferonni correction and/or Tukey’s multiple comparisons test. Mouse survival studies were statistically tested by the log-rank test. Tumor time course regressions were analyzed by mixed effect regression models. STATA v11.2 (StataCorp, College Station, TX) and Prism 6 (GraphPad Software, Inc. San Diego, CA) were utilized for statistical analyses and figure creation. Significance level of α ≤ 0.05 was set for all experiments. The dataset supporting the conclusions of this article is included within the article’s additional files [Additional file 3].
Systemic immune response
As was the case for the antibody concentration, the antigen-only vaccine elicited a moderate vaccine-specific CD8+ T-cell response that significantly differed from the mock vaccination by both percentage (p = 0.030; Fig. 1b) and total number (p < 0.001; Fig. 1c) of CD8+ T cells reactive to the immunogenic gp10025-33 peptide. The addition of MIP3α to the vaccine significantly increased the percentage of (p = 0.049) and total number of (p = 0.026) vaccine induced CD8+ T cells compared to the antigen only vaccine, increasing the CD8+ T cell numbers by 46% (Fig. 1b-c). The MIP3α-gp100 vaccine elicited significantly higher percentages and numbers of vaccine-specific CD8+ T cells compared to mock vaccination (p < 0.001 for both; Fig. 1b-c).
Therapeutic vaccination model
T-cell subset depletion
Tumor infiltrating lymphocytes
Our data demonstrate that the addition of the chemokine MIP3α to the gp100 DNA vaccine construct enhanced vaccine immunogenicity and therapeutic potential. Although the antigen-only vaccine elicited a significant anti-gp100 immune response compared to the mock vaccine, when utilized as a therapy, only the MIP3α-gp100 vaccine slowed tumor growth and enhanced mouse survival. Further, MIP3α fused to irrelevant antigen CSP showed no anti-tumor activity, despite the previously demonstrated ability of the CSP construct to function as a highly efficacious vaccine for preventing malaria in a mouse model system . Previous studies have conclusively shown that MIP3α must be fused to its antigen in order to enhance immunogenicity .
As has been shown in vitro [32, 33], MIP3α-gp100 vaccine directs the antigen in such a way that both CD4+ and CD8+ effector T-cells can be activated. In this study, T-cells were depleted after a prophylactic vaccination regimen in order to selectively deplete vaccine-specific effector cells and not disturb the immune activation phase of the vaccine response. If CD4+ T-cells were depleted in a therapeutic study, one would not know if the effect was due to lack of CD4+ anti-tumor effector response or due to lack of CD4+ T-cell help in the activation of a vaccine-specific CD8+ T-cell response.
Depletion of either the CD4+ or CD8+ effector T-cell population showed a protection phenotype similar to the non-depleted vaccine group, while depletion of both led to no protection, similar to that observed with mock vaccination. The lack of protection seen in the double depletion group provides evidence that antibodies elicited by the vaccine do not provide significant anti-tumor immunity on their own. Large tumor size outliers in both single depletion groups suggest that some proportion of the mice are reliant on the depleted subset for protection, but the overall groups either utilize both effector subsets relatively equally or one is able to compensate for lack of the other when necessary. The roles and mechanisms of tumor infiltrating effector CD4+ TILs are complex and still being defined , and therefore the intriguing finding of effector CD4+ T cells providing therapeutic efficacy in the absence of CD8+ T cells will be the subject of future work.
Finally, the data show that the therapeutic protection phenotype provided by MIP3α did not correlate with overall TILs, but did correlate with gp10025-33 vaccine peptide-reactive CD8+ TILs, elucidating that the immune activity of and not the quantity of the TILs correlates with therapeutic efficacy.
Vaccine efficacy depends on identification of appropriate target antigens, deliverance of those antigens in a form that elicits a relevant immune response, administration of vaccine by a route that brings it into contact with the critical immune cells, and selection of effective adjuvants/immunomodulators. For DNA vaccines, addition of MIP3α to circumsporozoite protein (CSP) with vaxfectin adjuvant  creates a robust, protective antibody response against malaria, addition of MIP3α to oncofetal antigen (OFA) given by gene gun creates a therapeutic response against lymphoma mediated by CD8+ T-cells , and as reported here, addition of MIP3α to gp100 given by intramuscular electroporation creates a therapeutic response against melanoma mediated by both CD4+ and CD8+ effector T-cells. All of these experiments have shown responses to be significantly enhanced by the chemokine in different contexts. Co-administration of MIP3α can enhance vaccine responses by enhanced DC recruitment . However, our previous studies have indicated that in the context of a DNA fusion vaccine, MIP3α is acting by directing nascent expressed protein antigens to DCs, not by recruiting DCs in vivo . Therefore, we hypothesize that in this context, the pro-inflammatory response elicited by electroporation serves as the adjuvant that recruits DCs to the vaccine site [19–21]. The MIP3α fused to gp100 then increases the efficiency of nascent vaccine protein uptake into infiltrating immature dendritic cells, resulting in enhanced downstream effector responses. This research provides further evidence for the utility of adding chemokine immunomodulators to vaccine constructs within any immunological context.
A primary strength of this DNA vaccine system is its modularity and ease of construction. This study shows that taking the gp100 antigen that induces a specific albeit not therapeutically relevant response on its own can become therapeutically relevant simply by fusing it to MIP3α. This observation raises the possibility that the response to more immunogenic antigens could be even further enhanced by the addition of MIP3α. A burgeoning new field in cancer vaccinology is the utilization of cancer-specific neoantigens as better vaccine targets that are not subject to T-cell central tolerance restrictions . Our modular DNA vaccine could easily and rapidly be constructed to utilize neoantigens as they are discovered in real time. Testing the principle of this idea will be the subject of future studies, utilizing now delineated immunogenic neoantigens found in the B16F10 cell line . In addition to neoantigens, future studies will also examine the efficacy of this vaccine system with other solid tumor models, in combination with current treatments such as immune checkpoint blockade, and in combination with novel immunomodulators.
In conclusion, our data show that addition of MIP3α enhances the immunogenicity and efficacy of a therapeutic vaccine against the aggressive solid tumor, B16F10 mouse melanoma. The addition of MIP3α to therapeutic vaccines could present a useful strategy to enhance the responses of currently studied vaccines. Furthermore, the modularity of the plasmid provides a realistic platform for creating neoantigen vaccines in a clinically relevant time frame. These findings show that MIP3α can be a plug and play addition to the cancer immunologist’s vaccine toolbox that deserves further testing to determine the true potential of this novel design.
Antigen presenting cell
Mouse melanoma model
Circumsporozoite protein from malaria-causing parasite Plasmodium falciparum
Dysfunctional Macrophage Inflammatory Protein-3α. When utilized as a vaccine with gp100, it can be referred to as ‘antigen-only vaccine’
Glycoprotein 100, common melanoma differentiation antigen and vaccine target
Major histocompatibility complex
Macrophage Inflammatory Protein-3α
We would like to acknowledge Dr. Fidel Zavala (Johns Hopkins School of Public Health, Baltimore, MD) for his generous gift of T-cell depletion antibodies. We would also like to acknowledge Dr. TC Wu (Johns Hopkins School of Medicine, Baltimore, MD) for allowing us to utilize his electroporator. We would like to acknowledge support for the statistical analysis from the National Center for Research Resources and the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health through Grant Number 1UL1TR001079, and more specifically Carol Thompson of the Johns Hopkins Biostatistics Center.
We have no funding sources to disclose.
Availability of data and materials
As reported in the Methods, the dataset supporting the conclusions of this article is included within the article’s additional files [Additional file 3].
JG performed, designed, and analyzed all the experiments and was the primary author of the manuscript. KL provided assisted with design and implementation of the mouse studies. HZ provided scientific direction, expertise, and help in data analysis. AB contributed to the conception and design of these experiments. RM contributed to the conception, design, analysis of data, and the writing of the manuscript. All authors read and approved the manuscript.
J. Gordy and R. Markham are inventors on pending patents using the vaccine platform described in this manuscript. R. Markham has equity interest in a company that has rights to this vaccine platform. The authors declare that they have no competing interests.
As reported in the Methods, mice were purchased from Charles River Laboratories (Wilmington, MA) and maintained in a pathogen-free micro-isolation facility in accordance with the National Institutes of Health guidelines for the humane use of laboratory animals. All experimental procedures involving mice were approved by the IACUC of the Johns Hopkins University (Protocol numbers MO13H219 and MO16H85).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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