Enhance Vaccine Efficacy Research Articles

CHO expressed recombinant human lactoferrin as an adjuvant for BCG.

Lactoferrin (LF), an iron binding protein with immune modulatory activities, has adjuvant activity to enhance vaccine efficacy. Tuberculosis (TB) is a pulmonary disease caused by the pathogen Mycobacterium tuberculosis (MTB). Progressive TB disease is clinically defined by damaging pulmonary pathology, a result of inflammation due to immune reactivity. The current vaccine for TB, an attenuated strain of Mycobacterium bovis, Bacillus Calmette Guerin (BCG), has only limited efficacy to prevent adult pulmonary TB. This study examines a Chinese hamster ovary (CHO) expressed recombinant human LF (rHLF) to boost efficacy of the BCG vaccine and delay early pathology post infectious challenge. C57BL/6 mice were immunized with BCG, or BCG admixed with either rHLF or bovine LF (bLF; internal control), or remained unvaccinated. Mice were then aerosol challenged with Erdman MTB. All vaccinated mice demonstrated decreased organ bacterial load up to 19 weeks post infection compared with non-vaccinated controls. Furthermore, mice receiving bLF or rHLF supplemented BCG vaccines showed a modest decrease in lung pathology developed over time, compared to the BCG vaccine alone. While mice vaccinated with BCG/rHLF demonstrated increased general lung inflammation at day 7, it occurred without noticeable increase in pro-inflammatory cytokines. At later times, decreased pathology in the rHLF groups correlated with decreased inflammatory cytokines. Splenic recall to BCG antigens showed BCG/rHLF vaccination increased production of IFN-γ, IL-6, and GM-CSF compared to naïve, BCG, and BCG/bLF groups. Analysis of T cell stimulating functions of bone marrow derived macrophages and dendritic cells treated with BCG/bLF or BCG/rHLF showed decreases in IL-10 production when co-cultured with sensitized CD4 and CD8 T cells, compared to those cultured with macrophages/dendritic cells treated with BCG without LF. These results indicate that addition of rHLF to the BCG vaccine can modulate development of host pathology early post infectious challenge, most likely through host immune regulation affecting hypersensitive responses.

Validation of alternative potency assays for influenza vaccines requires clinical studies

Readers of this Special Issue of Vaccine are likely unified by a desire to enhance vaccine efficacy and improve vaccine manufacturing efficiency. For influenza vaccines, challenges to achieving those goals are many, and range from improved surveillance to less cumbersome production platforms and more reliable performance verification. Specifically, demand is growing for an alternative vaccine potency assay. Assuming that a replacement potency assay is found to be promising, one question will be: how will it be judged to be accurate? It is generally agreed that any potential replacement for SRID will have to exhibit good correlation with SRID and yield a value within ±20% of the SRID determined potency. In my opinion, SRID itself has enough limitations that judging alternatives relative to that particular standard will not ensure that the industry as a whole transitions to an improved method. In fact, it could blind us to an assay that may ultimately provide a better predictive measure of vaccine efficacy (or immunogenicity). There is only one way to verify that measured potency is correlated with, and predictive of, measured immunogenicity – test the relationship in clinical studies.

Abstract CT201: The Mutanome Engineered RNA Immuno-Therapy (MERIT) project

Abstract The Mutanome Engineered RNA Immuno-Therapy (MERIT) consortium will clinically and industrially validate a pioneering RNA-based immunotherapy concept that targets individual tumor antigens and tumor-specific mutations in triple negative breast cancer (TNBC) patients. This biomarker-guided, personalized therapy is a collaborative effort of five partners from academia and industry and is funded by the European Commission's FP7 and led by BioNTech AG. TNBC is an aggressive, molecularly heterogeneous cancer that accounts for 20% of all breast cancer patients. The 5-year survival rate is less than 80%. The molecular heterogeneity across TNBCs results in a lack of common targetable molecular alterations, and thus targeted therapies frequently fail to provide clinical benefit. The MERIT concept attempts to address this unmet medical need. The personalized treatment consists in (i) injecting vaccines containing “off the shelf” mRNAs selected from a pre-synthesized mRNA vaccine warehouse (MERIT WAREHOUSE) that encode tumor specific antigens expressed in the respective patient's tumor; and (ii) thereafter mRNAs engineered on-demand that encode patient-specific sequence stretches incorporating non-synonymous mutations identified by next generation sequencing (NGS) and ranked by predicted immunogenicity (MERIT MUTANOME). The mRNAs are administered intravenously as a nanoparticulate lipoplex formulation and are selectively delivered to splenic APCs. The encoded antigens are translated into proteins that are rapidly processed. Subsequent peptide presentation on the surface of APCs induces antigen-specific T cell responses. The central part of the MERIT project, a multi-center first in human trial, will assess the feasibility, safety and biological efficacy of this innovative personalized immunotherapy in TNBC patients. After discussing the regulatory challenges with the German national regulatory agency (PEI), a phase I study is now in preparation. The trial will start in Q2 2015 in five academic centers in Europe and will recruit thirty TNBC patients. Furthermore, the project includes a comprehensive T-cell immunomonitoring and biomarker program. Moreover, an extensive research program will address the optimization of algorithms for improved prediction of immunogenic mutations. Additionally, compounds to enhance vaccine efficacy will be developed and improved to support further clinical development. We have established a RNA delivery platform as well as a MERIT WAREHOUSE containing mRNAs coding for a selection of TNBC specific antigens. Additionally, we have built a multi-disciplinary clinical workflow and trial design tailored to this unique therapeutic concept. We will describe the therapeutic concept and the critical skills, and methodologies required for this project, including cancer genomics, NGS, bioinformatics, tumor immunomics, industrial drug development, GMP manufacturing, clinical immunotherapy and immunological monitoring. Citation Format: Sandra Heesch, Cedrik M. Britten, Valesca Bukur, Janina Buck, John Castle, Jan Diekmann, Mustafa Diken, Katrin Frenzel, Sebastian Kreiter, Andreas N. Kuhn, Klaus Kuehlcke, Martin Loewer, Heinrich Haas, Alexandra Kemmer-Brueck, Bjoern-Philipp Kloke, Burkhard Otte, Anna Paruzynski, Sebastian Petri, Doreen Schwarck-Kokarakis, Marcus Schmidt, Fabrice André, Jacques De Greve, Thomas Kuendig, Henrik Lindman, Steve Pascolo, Tobias Sjöblom, Kris Thielemans, Laurence Zitvogel, Oezlem Tuereci, Ugur Sahin. The Mutanome Engineered RNA Immuno-Therapy (MERIT) project. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT201. doi:10.1158/1538-7445.AM2015-CT201

Controlled release of a model vaccine by nanoporous ceramic microneedle arrays

Current vaccination technology can advance from the use of novel ceramic nanoporous microneedle arrays (npMNA), where the material serves as a storage reservoir for vaccines. Moreover, npMNA will enhance vaccine efficacy by more precisely reaching skin dendritic cells, the kickstarters of T and B cell immunity. In the present study we assessed the efficacy of vaccination using npMNAs by in vivo application of OVA257-264 peptides mixed with agonistic anti-CD40 antibodies as adjuvant. The induction of OVA-specific CD8+ T cells via npMNA was comparable with the frequency induced via intradermal injection using needle-syringe. However, only when expanding the vaccination area by using two npMNAs the frequencies of induced IFN-γ-specific effector CD8+ T cells were comparable with those induced via needle-syringe injection. Analysis of vaccine release from npMNA in a human ex vivo skin explant model revealed that OVA257-264 peptides were indeed delivered intradermal, and release also increased by prolonging the npMNA application time on the human skin. Together, our studies demonstrate the potential of npMNA for vaccine delivery in human skin and in vivo induction of CD8+ effector T cell responses.

Novel synthetic plasmid and Doggybone™ DNA vaccines induce neutralizing antibodies and provide protection from lethal influenza challenge in mice

Nucleic acid-based vaccines (NAVs) are a promising alternative to conventional influenza vaccines with the potential to increase influenza vaccine availability due to their simplicity in design and rapid speed of production. NAVs can also target multiple influenza antigens and control flu variants. Traditionally NAVs have been DNA plasmids however, we are continuing to explore new methods that may enhance vaccine efficacy. Recently new focus has been on RNA cassettes as NAVs. RNA vaccines combine conceptual advantages in that they focus on delivery of only the coding cassette. However, RNA vaccines have a short half-life and cause interferon-induced fevers. Here we describe a new NAV approach where we study delivery of a linear DNA cassette [Doggybone™ linear closed DNA [(dbDNA™)] produced by an enzymatic process that yields an antigen expression cassette comprising a promoter, DNA antigen, poly A tail, and telomeric ends. This focused approach has many of the advantages of plasmid DNA as well as a minimal cassette size similar to RNA strategies. For this study, we characterized the specific CD4+ and CD8+ T cell responses and determined the hemagglutination inhibition (HI) titers induced by dbDNA™ and compared the responses with those of an optimized plasmid DNA (pDNA) vaccine encoding the same H1N1 influenza A/PR/8/34 HA gene. Immunizations with the constructs resulted in similar humoral and cellular immune responses. Both constructs induced high-titer HI antibodies and fully protected animals from lethal viral challenge. The data obtained from this study provides important validation for further development of novel vector approaches.

Natural killer cells suppress B cell immunity through perforin-dependent mechanisms (IRC4P.617)

Abstract Accumulating evidence suggests that natural killer (NK) cells are critical regulators of antiviral T cells during chronic infection. We find that NK cells are also potent inhibitors of germinal center (GC) responses and the generation of long-lived B cell immunity after acute infection. A similar suppressive function was noted for NK cells after immunization with the non-replicating T cell-dependent antigen NP-KLH, suggesting that strategies to subvert NK cell-mediated inhibition may be able to improve vaccine responses. To determine whether cytotoxic function is important for this regulatory activity, we examined the contributions of perforin-containing granules and the death receptor Fas. NK cell inhibition of GCs was intact in Fas-deficient lpr mice but absent in perforin-deficient mice, thereby implicating granule-mediated cytotoxicity in this process. Moreover, we found that NK cells acted in a perforin-dependent manner during the first three days post-immunization to suppress generation of follicular helper CD4 T cell (Tfh) responses. The suppression of Tfh by NK cells was independent of the presence or absence of B cells, while CD4 T cells were required for development of virus-specific B cells. Thus, NK cells determine the quality and magnitude of B cell responses following immunization predominately via perforin-dependent repression of Tfh responses. This immunoregulatory process represents an innovative target for interventions aimed at enhancing vaccine efficacy.

MTORC1 and mTORC2 selectively regulate CD8⁺ T cell differentiation.

Activation of mTOR-dependent pathways regulates the specification and differentiation of CD4+ T effector cell subsets. Herein, we show that mTOR complex 1 (mTORC1) and mTORC2 have distinct roles in the generation of CD8+ T cell effector and memory populations. Evaluation of mice with a T cell-specific deletion of the gene encoding the negative regulator of mTORC1, tuberous sclerosis complex 2 (TSC2), resulted in the generation of highly glycolytic and potent effector CD8+ T cells; however, due to constitutive mTORC1 activation, these cells retained a terminally differentiated effector phenotype and were incapable of transitioning into a memory state. In contrast, CD8+ T cells deficient in mTORC1 activity due to loss of RAS homolog enriched in brain (RHEB) failed to differentiate into effector cells but retained memory characteristics, such as surface marker expression, a lower metabolic rate, and increased longevity. However, these RHEB-deficient memory-like T cells failed to generate recall responses as the result of metabolic defects. While mTORC1 influenced CD8+ T cell effector responses, mTORC2 activity regulated CD8+ T cell memory. mTORC2 inhibition resulted in metabolic reprogramming, which enhanced the generation of CD8+ memory cells. Overall, these results define specific roles for mTORC1 and mTORC2 that link metabolism and CD8+ T cell effector and memory generation and suggest that these functions have the potential to be targeted for enhancing vaccine efficacy and antitumor immunity.

A Brucella melitensis M5-90 wboA deletion strain is attenuated and enhances vaccine efficacy

Brucella spp. are Gram-negative intracellular pathogens of both humans and animals that cause great economic burdens in developing countries. Live attenuated vaccines are the most efficient means for the prevention and control of animal Brucellosis. However, Brucella vaccines (strain M5-90 and others) have several drawbacks and do not allow serological differentiation between vaccinated and infected animals. A wboA mutant was derived from Brucella melitensis (B. melitensis) vaccine strain M5-90 and tested for virulence and protective efficiency. T-cell responses (CD4+, CD8+), levels of immunoglobulin G (IgG), and cytokine production were observed. WboA was also assessed as a diagnostic marker for Brucellosis. B. melitensis strain M5-90ΔwboA exhibited reduced survival in murine macrophages (RAW 264.7) and BALB/c mice and induced protective immunity in mice comparable to that from the parental strain M5-90. In mice, the wboA mutant elicited an anti-Brucella-specific IgG response and induced the secretion of gamma interferon (IFN-γ) and interleukin-2 (IL-2). In sheep, M5-90ΔwboA immunization induced the secretion of IFN-γ, and serum samples from sheep inoculated with M5-90ΔwboA were negative by Bengal Plate Test (RBPT) and Standard Tube Agglutination Test (STAT). In mice, probes against WboA antigen allowed for serological differentiation between natural infection and vaccination. The M5-90ΔwboA mutant is a potential attenuated live vaccine candidate against virulent B. melitensis 16M infection. It will be further evaluated in livestock.

Lactobacillus rhamnosus GG ATCC 53103 (LGG) as an Immune Adjuvant for Influenza Vaccination in the Elderly

Annual vaccination against seasonal influenza in the U.S. is recommended for all persons aged 蠅 6 months. A moderate effectiveness for influenza vaccines of approximately 60% has been estimated. Little is known about mechanisms needed to enhance vaccine efficacy against influenza. We investigated the effect of feeding LGG to clinically healthy elderly subjects participating in two separate randomized double‐blind placebo‐controlled studies (RCT) where subjects consumed 2 x 1010 cfu of LGG or placebo for 28 days after vaccination with either Trivalent Inactivated Influenza vaccine (TIV) (n=27), or Live Attenuated Influenza Vaccine (LAIV)(n=30). Transcriptome among treatment groups was compared in each RCT using deep RNA sequencing (RNA‐seq) of 500ng of RNA isolated from whole blood of all subjects prior (baseline) during (day 21 and 28), and 28 days after feeding of LGG had ceased (day 56). Differentially expressed genes (DEG) with an absolute fold difference >1.5 were analyzed after data mining using Ingenuity Pathway Analysis. Enrichment of genes associated with inflammation and production of IFNs were seen in LGG‐treated subjects with LAIV vaccination after 21 and 28 days of continuous LGG treatment. No significant changes were seen after LGG feeding ceased in LAIV vaccinated patients or in any of the subjects given TIV vaccination. Measuring DEG by RNA‐seq can be used to evaluate immune adjuvant properties of LGG with different vaccine preparations.

Enhancement of protective efficacy through adenoviral vectored vaccine priming and protein boosting strategy encoding triosephosphate isomerase (SjTPI) against Schistosoma japonicum in mice.

BackgroundSchistosomiasis japonica is a zoonotic parasitic disease; developing transmission blocking veterinary vaccines are urgently needed for the prevention and control of schistosomiasis in China. Heterologous prime-boost strategy, a novel vaccination approach, is more effective in enhancing vaccine efficacy against multiple pathogens. In the present study, we established a novel heterologous prime-boost vaccination strategy, the rAdV-SjTPI.opt intramuscular priming and rSjTPI subcutaneous boosting strategy, and evaluated its protective efficacy against Schistosoma japonicum in mice.Methodology/Principal FindingsAdenoviral vectored vaccine (rAdV-SjTPI.opt) and recombinant protein vaccine (rSjTPI) were prepared and used in different combinations as vaccines in a mouse model. The specific immune responses and protective efficacies were evaluated. Furthermore, the longevity of protective efficacy was also determined. Results showed that the rAdV-SjTPI.opt priming-rSjTPI boosting strategy elicited higher levels of specific IgG responses and broad-spectrum specific cellular immune responses. The protective efficacy could reach up to nearly 70% and 50% of protection could be observed at 10 weeks after the last immunization in mice.Conclusions/SignificanceThe rAdV-SjTPI.opt intramuscular priming-rSjTPI subcutaneous boosting vaccination strategy is a novel, highly efficient, and stable approach to developing vaccines against Schistosoma japonicum infections in China.

Targeting myeloid cells in the tumor microenvironment enhances vaccine efficacy in murine epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is typically diagnosed at advanced stages, and is associated with a high relapse rate. Patients in remission are ideal candidates for immunotherapy aimed at cure or prolonging disease-free periods. However, immunosuppressive pathways in the tumor microenvironment are obstacles to durable anti-tumor immunity. In a metastatic syngeneic mouse model of EOC, immunosuppressive macrophages and myeloid-derived suppressor cells (MDSCs) accumulate in the local tumor environment. In addition, resident peritoneal macrophages from non-tumor-bearing mice were highly immunosuppressive, abrogating stimulated T cell proliferation in a cell contact-dependent manner. Immunization with microparticles containing TLR9 and NOD-2 ligands (MIS416) significantly prolonged survival in tumor-bearing mice. The strategy of MIS416 immunization followed by anti-CD11b administration further delayed tumor progression, thereby establishing the proof of principle that myeloid depletion can enhance vaccine efficacy. In patients with advanced EOC, ascites analysis showed substantial heterogeneity in the relative proportions of myeloid subsets and their immunosuppressive properties. Together, these findings point to immunosuppressive myeloid cells in the EOC microenvironment as targets to enhance vaccination. Further studies of myeloid cell accumulation and functional phenotypes in the EOC microenvironment may identify patients who are likely to benefit from vaccination combined with approaches that deplete tumor-associated myeloid cells.

Cross-presentation through langerin and DC-SIGN targeting requires different formulations of glycan-modified antigens

Dendritic cells (DCs) and Langerhans cells (LC) are professional antigen presenting cells (APCs) that initiate humoral and cellular immune responses. Targeted delivery of antigen towards DC- or LC-specific receptors enhances vaccine efficacy. In this study, we compared the efficiency of glycan-based antigen targeting to both the human DC-specific C-type lectin receptor (CLR) DC-SIGN and the LC-specific CLR langerin. Since DC-SIGN and langerin are able to recognize the difucosylated oligosaccharide Lewis Y (LeY), we prepared neoglycoconjugates bearing this glycan epitope to allow targeting of both lectins. LeY-modified liposomes, with an approximate diameter of 200nm, were significantly endocytosed by DC-SIGN+ DCs and mediated efficient antigen presentation to CD4+ and CD8+ T cells. Surprisingly, although langerin bound to LeY-modified liposomes, LCs exposed to LeY-modified liposomes could not endocytose liposomes nor mediate antigen presentation to T cells. However, LCs mediated an enhanced cross-presentation when antigen was delivered through langerin using LeY-modified synthetic long peptides. In contrast, LeY-modified synthetic long peptides were recognized by DC-SIGN, but did not trigger antigen internalization nor antigen cross-presentation. These data demonstrate that langerin and DC-SIGN have different size requirements for antigen uptake. Although using glycans remains an interesting option in the design of anti-cancer vaccines targeting multiple CLRs, aspects such as molecule size and conformation need to be taken in consideration.

Poly(lactic acid) and poly(lactic-co-glycolic acid) particles as versatile carrier platforms for vaccine delivery.

The development of safe and effective vaccines for cancer and infectious diseases remains a major goal in public health. Over the last two decades, controlled release of vaccine antigens and immunostimulant molecules has been achieved using nanometer or micron-sized delivery vehicles synthesized using biodegradable polymers. In addition to achieving a depot effect, enhanced vaccine efficacy using such delivery vehicles has been attributed to efficient targeting of antigen presenting cells such as dendritic cells. Biodegradable and biocompatible poly(lactic acid) and poly(lactic-co-glycolic acid) polymers belong to one such family of polymers that have been a popular choice of material used in the design of these delivery vehicles. This review summarizes research findings from ourselves and others highlighting the promise of poly(lactic acid)- and poly(lactic-co-glycolic acid)-based vaccine carriers in enhancing immune responses.

PH-sensitive carbonate apatite nanoparticles as DNA vaccine carriers enhance humoral and cellular immunity

To demonstrate the potential of pH-sensitive carbonate apatite (CO3Ap) nanoparticles as DNA vaccine carriers to enhance vaccination efficacy, we examined the humoral and cellular immune responses of C57BL/6 mice immunized with the plasmid expression vector pCI-neo encoding the full-length soluble ovalbumin (OVA) (pCI-neo-sOVA), pCI-neo-sOVA/CO3Ap complexes, or pCI-neo/CO3Ap complexes as a control. Mice immunized with a low dose of pCI-neo-sOVA-loaded CO3Ap (10μg) produced ex vivo splenocyte proliferation after stimulation with CD8 T-cell but not CD4 T-cell epitopes and a delayed-type-hypersensitivity reaction more efficiently than mice in the other groups. Furthermore, mice receiving this immunization generated the same levels of OVA-specific antibodies and interferon (IFN)-γ secretion after CD8 T-cell and CD4 T-cell epitope challenges as those in mice treated with 100μg of free pCI-neo-sOVA, whereas mice injected with a high dose of pCI-neo-sOVA-loaded CO3Ap (100μg) or with control plasmids produced negligible levels of OVA-specific antibodies or IFN-γ. Therefore, our results showed that 10μg of pCI-neo-sOVA delivered by CO3Ap strongly elicited humoral and cellular immune responses. This study is the first to demonstrate the promising potential of CO3Ap nanoparticles for DNA vaccine delivery.

A protective role of murine langerin⁺ cells in immune responses to cutaneous vaccination with microneedle patches.

Cutaneous vaccination with microneedle patches offers several advantages over more frequently used approaches for vaccine delivery, including improved protective immunity. However, the involvement of specific APC subsets and their contribution to the induction of immunity following cutaneous vaccine delivery is not well understood. A better understanding of the functions of individual APC subsets in the skin will allow us to target specific skin cell populations in order to further enhance vaccine efficacy. Here we use a Langerin-EGFP-DTR knock-in mouse model to determine the contribution of langerin+ subsets of skin APCs in the induction of adaptive immune responses following cutaneous microneedle delivery of influenza vaccine. Depletion of langerin+ cells prior to vaccination resulted in substantial impairment of both Th1 and Th2 responses, and decreased post-challenge survival rates, in mice vaccinated cutaneously but not in those vaccinated via the intramuscular route or in non-depleted control mice. Our results indicate that langerin+ cells contribute significantly to the induction of protective immune responses following cutaneous vaccination with a subunit influenza vaccine.

Aptamer-targeted Antigen Delivery

Effective therapeutic vaccines often require activation of T cell-mediated immunity. Robust T cell activation, including CD8 T cell responses, can be achieved using antibodies or antibody fragments to direct antigens of interest to professional antigen presenting cells. This approach represents an important advance in enhancing vaccine efficacy. Nucleic acid aptamers present a promising alternative to protein-based targeting approaches. We have selected aptamers that specifically bind the murine receptor, DEC205, a C-type lectin expressed predominantly on the surface of CD8α+ dendritic cells (DCs) that has been shown to be efficient at facilitating antigen crosspresentation and subsequent CD8+ T cell activation. Using a minimized aptamer conjugated to the model antigen ovalbumin (OVA), DEC205-targeted antigen crosspresentation was verified in vitro and in vivo by proliferation and cytokine production by primary murine CD8+ T cells expressing a T cell receptor specific for the major histocompatibility complex (MHC) I-restricted OVA257–264 peptide SIINFEKL. Compared with a nonspecific ribonucleic acid (RNA) of similar length, DEC205 aptamer-OVA-mediated antigen delivery stimulated strong proliferation and production of interferon (IFN)-γ and interleukin (IL)-2. The immune responses elicited by aptamer-OVA conjugates were sufficient to inhibit the growth of established OVA-expressing B16 tumor cells. Our results demonstrate a new application of aptamer technology for the development of effective T cell-mediated vaccines.

Targeting myeloid cells in the tumor microenvironment as a strategy to enhance vaccine efficacy in epithelial ovarian cancer

Objectives: Ovarian cancer vaccine development is hampered by the availability of powerful immune adjuvants that can induce sustained innate immunity and augment adaptive cellular responses in the tumor-bearing host. Accumulation of myeloid cells in the tumor microenvironment is a known obstacle to durable antitumor immunity. We evaluated the strategy of vaccination with the novel TLR9/NOD2 ligand (MIS-416) followed by myeloid cell depletion in the murine model of epithelial ovarian cancer (EOC).

Elimination of IL-10-inducing T-helper epitopes from an IGFBP-2 vaccine ensures potent antitumor activity.

Immunization against self-tumor antigens can induce T-regulatory cells, which inhibit proliferation of type I CD4(+) T-helper (TH1) and CD8(+) cytotoxic T cells. Type I T cells are required for potent antitumor immunity. We questioned whether immunosuppressive epitopes could be identified and deleted from a cancer vaccine targeting insulin-like growth factor-binding protein (IGFBP-2) and enhance vaccine efficacy. Screening breast cancer patient lymphocytes with IFN-γ and interleukin (IL)-10 ELISPOT, we found epitopes in the N-terminus of IGFBP-2 that elicited predominantly TH1 whereas the C-terminus stimulated TH2 and mixed TH1/TH2 responses. Epitope-specific TH2 demonstrated a higher functional avidity for antigen than epitopes, which induced IFN-γ (P = 0.014). We immunized TgMMTV-neu mice with DNA constructs encoding IGFBP-2 N-and C-termini. T cell lines expanded from the C-terminus vaccinated animals secreted significantly more type II cytokines than those vaccinated with the N-terminus and could not control tumor growth when infused into tumor-bearing animals. In contrast, N-terminus epitope-specific T cells secreted TH1 cytokines and significantly inhibited tumor growth, as compared with naïve T cells, when adoptively transferred (P = 0.005). To determine whether removal of TH2-inducing epitopes had any effect on the vaccinated antitumor response, we immunized mice with the N-terminus, C-terminus, and a mix of equivalent concentrations of both vaccines. The N-terminus vaccine significantly inhibited tumor growth (P < 0.001) as compared with the C-terminus vaccine, which had no antitumor effect. Mixing the C-terminus with the N-terminus vaccine abrogated the antitumor response of the N-terminus vaccine alone. The clinical efficacy of cancer vaccines targeting self-tumor antigens may be greatly improved by identification and removal of immunosuppressive epitopes.

Heat shock protein vaccination and directed IL-2 therapy amplify tumor immunity rapidly following bone marrow transplantation in mice

AbstractTumor relapse is the primary cause of mortality in patients with hematologic cancers following autologous hematopoietic stem cell transplantation (HSCT). Vaccination early after HSCT can exploit both the state of lymphopenia and minimal residual disease for generating antitumor immunity. Here, multiple vaccinations using lymphoma cells engineered to secrete heat shock protein fusion gp96-Ig within 2 weeks of T cell-replete syngeneic HSCT led to cross-presentation and increased survival of lymphoma-bearing mice. To enhance vaccine efficacy, interleukin (IL)-2 was directed to predominantly memory phenotype CD8+ T lymphocytes and natural killer (NK) cells via administration bound to anti-IL-2 monoclonal antibody clone S4B6 (IL-2S4B6). Combination therapy with gp96-Ig vaccination and coordinated infusions of IL-2S4B6 resulted in marked prolongation of survival, which directly correlated with ∼500% increase in effector CD8+ T-cell numbers. Notably, this dual regimen elicited large increases in both donor CD8+ T and NK cells, but not CD4+ T lymphocytes; the former 2 populations are essential for both vaccine efficacy and protection against opportunistic infections after HSCT. Indeed, IL-2S4B6-treated HSCT recipients infected with Listeria monocytogenes exhibited decreased bacterial levels. These preclinical studies validate a new strategy particularly well suited to the post-HSCT environment, which may augment adaptive and innate immune function in patients with malignant disease receiving autologous HSCT.

Toll-Like Receptor 7 Agonist Imiquimod in Combination with Influenza Vaccine Expedites and Augments Humoral Immune Responses against Influenza A(H1N1)pdm09 Virus Infection in BALB/c Mice

Toll-like receptors (TLRs) of the innate immune system are known targets for enhancing vaccine efficacy. We investigated whether imiquimod, a synthetic TLR7 agonist, can expedite the immune response against influenza virus infection when combined with influenza vaccine. BALB/c mice were immunized intraperitoneally with monovalent A(H1N1)pdm09 vaccine combined with imiquimod (VCI) prior to intranasal inoculation with a lethal dose of mouse-adapted A(H1N1)pdm09 virus. For mice immunized 3 days before infection, the survival rates were significantly higher in the VCI group (60%, mean survival time[MST], 11 days) than in the vaccine-alone (30%; MST, 8.8 days), imiquimod-alone (5%; MST, 8.4 days), and phosphate-buffered saline (PBS) (0%; MST, 6.2 days) groups (P < 0.01). In the VCI group, 45 and 35% of the mice survived even when they were infected 2 days or 1 day after immunization. Virus-specific serum IgM, IgG, and neutralizing antibodies appeared earlier with higher geometric mean titers in the VCI group than in the control groups. The pulmonary viral load was significantly lower at all time points postinfection in the VCI, vaccine-alone, and imiquimod-alone groups than in the PBS control group (P < 0.05). The protection induced by VCI was specific for A(H1N1)pdm09 virus but not for A(H5N1) virus. Since imiquimod combined with RNase-treated vaccine is as protective as imiquimod combined with untreated vaccine, mechanisms other than TLR7 may operate in expediting and augmenting immune protection. Moreover, increased gamma interferon mRNA expression and IgG isotype switching, which are markers of the Th1 response induced by imiquimod, were not apparent in our mouse model. The mechanisms of imiquimod-induced immune protection deserve further study.