Combination of nanoparticle-based therapeutic vaccination and transient ablation of regulatory T cells enhances anti-viral immunity during chronic retroviral infection
© Knuschke et al. 2016
Received: 16 February 2016
Accepted: 28 March 2016
Published: 14 April 2016
Regulatory T cells (Tregs) have been shown to limit anti-viral immunity during chronic retroviral infection and to restrict vaccine-induced T cell responses. The objective of the study was to assess whether a combinational therapy of nanoparticle-based therapeutic vaccination and concomitant transient ablation of Tregs augments anti-viral immunity and improves virus control in chronically retrovirus-infected mice. Therefore, chronically Friend retrovirus (FV)-infected mice were immunized with calcium phosphate (CaP) nanoparticles functionalized with TLR9 ligand CpG and CD8+ or CD4+ T cell epitope peptides (GagL85–93 or Env gp70123–141) of FV. In addition, Tregs were ablated during the immunization process. Reactivation of CD4+ and CD8+ effector T cells was analysed and the viral loads were determined.
Therapeutic vaccination of chronically FV-infected mice with functionalized CaP nanoparticles transiently reactivated cytotoxic CD8+ T cells and significantly reduced the viral loads. Transient ablation of Tregs during nanoparticle-based therapeutic vaccination strongly enhanced anti-viral immunity and further decreased viral burden.
Our data illustrate a crucial role for CD4+ Foxp3+ Tregs in the suppression of anti-viral T cell responses during therapeutic vaccination against chronic retroviral infection. Thus, the combination of transient Treg ablation and therapeutic nanoparticle-based vaccination confers robust and sustained anti-viral immunity.
Effector T cells are crucial for the elimination of intracellular pathogens. Especially cytotoxic CD8+ T cells are predominantly important for the control of virus infections, i.e. human immunodeficiency virus (HIV)  or hepatitis C virus (HCV) [2, 3]. However, several evasion mechanisms of viruses, like viral evolution or exhaustion of effector T cells can contribute to persistent infection. Important inducer of dysfunctional CD8+ T cells are virus-induced or virus-expanded regulatory T cells (Tregs). Tregs belong to a specialized T cell subset which is characterized by the expression of the transcription factor Foxp3 and unique suppressive capacities that can limit anti-viral immune responses [4–6]. It was demonstrated that the low responsiveness and reduced proliferation of virus-specific T cells during chronic viral infection is associated with the expansion of Tregs which also affects the cytotoxic activity of virus-specific CD8+ T cells [6, 7]. Immune-based therapies have been considered as potent approach to efficiently reactivate impaired anti-viral effector immunity to control chronic viral infection. In this concern, depletion of Tregs during acute and chronic murine retroviral infection was shown to restore the functions of CD8+ T cells and to decrease viral loads [5, 6, 8, 9]. The results suggest that Treg-mediated immunosuppression is a significant factor in the maintenance of chronic viral infections, and that Treg-targeted immunotherapy could be a valuable component in therapeutic strategies to treat chronic retroviral infections.
Additionally, promising trials to efficiently reactivate CD8+ T cell responses have been made by confronting patient-derived dendritic cells (DCs) with HIV-specific antigens to induce virus-specific immune responses [10, 11] or by directly targeting lymph nodes with peptide-based vaccine vehicles . In this context, nanoparticulate structures are discussed to be ideal vaccine delivery vehicles . Inorganic calcium phosphate (CaP) nanoparticles are promising candidates for this purpose. Due to their small size (~100 nm) and their non-toxicity CaP nanoparticles feature ideal properties as delivery vehicles across cell membranes [14, 15]. After functionalization with TLR ligands such as CpG or poly(I:C), CaP nanoparticles efficiently activate cells of the innate and adaptive immune system . In a recent study, we have shown that prophylactic immunization of mice with CaP nanoparticles, functionalized with virus-derived T cell epitopes and the TLR9 ligand CpG, leads to an activation of DCs and in consequence induced strong antigen-specific CD4+ and CD8+ T cell response. Of note, this vaccination strategy resulted in a powerful protection against influenza virus and Friend retrovirus (FV) infection [16, 17]. In addition, therapeutic vaccination of chronically FV-infected mice efficiently reactivated effector T cells which led to a significant decrease in viral loads.
FV is an immunosuppressive retrovirus complex of the non-pathogenic Friend murine leukemia virus (F-MuLV) and the pathogenic, replication defective spleen focus forming virus (SFFV) . Disease-resistant mouse strains can control acute infection, but develop a life-long chronic infection. This is associated with the expansion and activation of Tregs , which down-regulate virus-specific cytotoxic CD8+ T cell activity , a conspicuous feature which in fact was also seen in HIV infection . As no appropriate mouse model for studying HIV infection exists, the well-described FV infection model has been accepted to be suitable to understand basic concepts in retroviral immunity and to test novel vaccination strategies [21–24].
In this study, we aimed to identify whether nanoparticle-based vaccination and concomitant modulation of inhibitory Treg responses in chronically retrovirus infected mice results in potent anti-viral immunity and improves viral control. To our knowledge, we show for the first time that the combinational immune-based therapy of CaP nanoparticle vaccination and Treg ablation efficiently reactivates the cytotoxic potential of CD8+ effector T cells and leads to a dramatic drop in viral loads in chronic retroviral infection, stronger than each therapeutic treatment regimen alone. Thus, vaccine-induced reactivation of effector T cells during chronic viral infection is feasible but is strikingly enhanced by simultaneous immune modulation.
Therapeutic vaccination of chronically infected mice with functionalized CaP nanoparticles transiently reactivates cytotoxic CD8+ T cells and reduce viral loads
Recently, we introduced functionalized CaP nanoparticles as excellent vaccination delivery system for the treatment of chronic retroviral infection . Functionalized triple-shell CaP nanoparticles were prepared by subsequent precipitation and functionalization steps as described earlier [14, 16]. In brief, CaP solid cores were loaded with CpG and CD8+ or CD4+ T cell epitope peptides (GagL85–93 or Env gp70123–141) of FV and finally coated with a second layer of CaP and an outer shell of CpG for colloidal stabilization (Additional file 1: Figure S1A). Vaccination of mice with functionalized CaP nanoparticles efficiently activates the major DC subsets, therefore mediating ideal requirements for the initiation of adaptive immune responses [14, 16]. One shot-immunization of chronically FV infected mice with functionalized CaP nanoparticles reactivated virus-specific T cell responses accompanied by a significant decrease in viral loads with striking advantage over vaccination with soluble CpG ligand and viral peptides  (Additional file 1: Figure S1B–E).
Foxp3+ CD4+ regulatory T cells down-regulate T cell responses after CaP nanoparticle vaccination
Transient ablation of regulatory T cells strongly enhances the therapeutic effect of functionalized CaP nanoparticles
Antagonizing chemokine receptor 4 on regulatory T cells does not improve the anti-viral effect of therapeutic vaccination with functionalized CaP nanoparticles
Depletion of regulatory T cells in humans, as performed in the transgenic DEREG mouse model, is still elusive. Regulatory T cell depletion by anti-CD25 monoclonal antibodies can lead to enhanced immunity; however, a CD25-targeted approach is associated with several drawbacks. First, regulatory T cell depletion might lead to appearance of autoimmune manifestations, as reported in experimental animals. Secondly, activated effector CD4+ and CD8+ T cells also express CD25 and could also be eliminated by CD25 depletion . Therefore, alternative approaches, focusing on inhibition instead of depletion of regulatory T cells might be of high interest for upcoming therapies.
Viruses such as HBV or HIV possess the ability to evade from the immune system by several mechanisms, like viral evolution or exhaustion of effector T cells which can lead to persistent infection. The currently available treatments for many of these chronic infections do not lead to satisfactory results. For example antiretroviral therapy (ART) is able to suppress HIV replication, the most prominent member of the retrovirus family, but the fact that HIV persists in reservoirs prevents HIV cure by ART. Therefore, there is a strong need to develop new strategies for therapeutic vaccination against chronic infection. Nanomaterials are discussed as part of potential immune-based therapeutic treatment to reactivate the host’s immune response [12, 30]. In our latest report, we demonstrated that the application of CpG and viral peptide functionalized CaP nanoparticles leads to significant reactivation of T cell responses and improves virus control in murine chronic FV infection . We also noticed that Tregs have a strong impact on virus-specific immune responses during chronic retrovirus infection . They seem to have a significant effect on the cytotoxicity of CD8+ T cells during acute chronic FV infection by inhibiting the production of cytotoxic molecules such as granzyme A and B . Thus, the aim of the current study was to determine whether the combinational therapy of nanoparticle-based vaccination with depletion of Tregs could strongly enhance the cytotoxic T cell (CTL) response and opens new options in the fight against chronic retroviral infection.
Our current study demonstrates that a combination of depletion of immunosuppressive Tregs and therapeutic immunization with functionalized CaP nanoparticles of chronically retrovirus infected mice significantly reduced viral loads by efficiently reactivating the cytotoxic potential of virus-specific CD8+ and CD4+ effector T cells compared to therapeutic vaccination alone. It therefore underlines the considerable influence of Tregs on the effector T cell response during immunotherapy which should be considered for the development of new vaccination strategies.
Tregs are a subset of CD4+ T lymphocytes with the ability to down-regulate the immune system . They are the key modulators of the establishment and/or maintenance of viral chronicity and constitute a barrier to efficient vaccination and immunotherapeutic strategies . The implication of regulatory T cells in chronic viral infection was first described in mice infected with FV [33, 34] and was then extended to other persistent viruses, including HIV , HBV , and HCV . Especially for HIV patients, it was shown that Tregs similarly to the situation in chronic FV infection accumulate in lymphoid tissues and suppress the anti-HIV response [35, 37]. Thus, it is becoming increasingly clear that controlling this immunosuppressive cell subset would have widespread clinical applications to fight life-threatening viral diseases . In addition, our current study shows evidence that the Treg population also counteracts the effects of immunotherapeutic vaccination which implies that controlling this subset could further enhance immunotherapy. Suvas et al.  have shown that treatment of mice with anti-CD25 antibodies to impair the CD25high expressing Treg population resulted in enhanced antigen-specific CD8+ T-cell responses accompanied by reduced viral burden in herpes simplex virus-1 infected mice. In addition, we could demonstrate in recent studies that ablation of Tregs from mCMV- and FV-infected mice by using DEREG (DEpletion of Treg) mice was associated with elevated anti-viral CD8+ T-cell responses showing a restored functionality [5, 40]. Also human studies have highlighted the effects of Tregs in suppressing antiviral effector responses. Macatangay et al.  showed that reduced frequencies of virus-specific Tregs during vaccination are important for an efficient therapeutic anti-HIV vaccine since this enables the broad activation of effector T cells. Furthermore, an decrease HIV-specific Treg response was associated with an effective DC-vaccine induced immunity in HIV-infected patients .
However, the depletion of Tregs should be done with caution since it is known that long term depletion of Tregs can lead to severe autoimmunity syndromes . Temporary Treg depletion or the addition of a Treg blocker along with the vaccine should be considered. Particularly, transient inhibition of Treg migration by using small molecule antagonists to CCR4 was shown to provide robust antigen-specific CD8+ T cell responses during anti-tumor vaccination [28, 29]. However, treatment of chronically FV-infected mice with a CCR4 antagonist before and/or during vaccination with functionalized CaP nanoparticles within our experimental setting did not improve the vaccination success. Therefore, further studies are needed to elucidate how to manipulate Treg numbers or function during anti-viral vaccination.
Several lines of evidence suggest now that the depletion of Tregs during therapeutic immunization of chronically retrovirus infected mice improves the vaccination efficacy of functionalized CaP nanoparticles. First, depletion of Tregs during immunization enhanced the relative number of virus-specific IFN-γ producing CD8+ T cells. Second, the strongest increase in the frequency of total activated virus-specific cytotoxic CD8+ CD43+ GzmB+ T cells was observed after a combined depletion of Tregs and immunization. Since granzyme B has been associated with virus control and killing of virus infected cells in both FV and HIV infection [44, 45], induction of cytotoxic T cells is crucial for a therapeutic vaccine. Finally, depletion of Tregs during the therapeutic immunization of chronically FV-infected mice resulted in the lowest viral loads compared to all other treatments tested in this study. Thus Tregs clearly control the CTL response induced by therapeutic vaccination and may reduce its effect.
Our findings underline that a combination of Treg ablation and therapeutic nanoparticle-based vaccination confers robust antiviral immunity and a sustained control of chronic virus infection.
C57BL/6 mice were purchased from Harlan Laboratories (Harlan Winkelmann GmbH, Borchen, Germany). DEREG (DEpletion of REGulatory T cells) mice (expressing eGFP and diphtheria toxin receptor under the control of the forkhead box P3 [Foxp3] promoter) on C57BL/6 background were described previously . All mice used in the experiments were 8–10 weeks old at time point of infection.
Cells and cell culture
A murine fibroblast cell line from Mus dunni was maintained in Roswell Park Memorial Institute (RPMI) medium containing 10 % FCS and 50 µg mL−1 penicillin/streptomycin. Cell lines were maintained in a humidified 5 % CO2 atmosphere at 37 °C.
Preparation of functionalized nanoparticles
CpG and viral peptide functionalized multi shell CaP nanoparticles were prepared as described previously . Briefly, nanoparticles were prepared by fast mixing of aqueous solutions of calcium nitrate (6.25 mM) and diammonium hydrogen phosphate (3.74 mM). Immediately after mixing, 1 mL of the calcium phosphate nanoparticle dispersion was mixed with 0.2 mL CpG (63 µM = 400 µg mL−1) and 0.05 mL of the viral peptides GagL85–93 and gp70123–141 (1 mg mL−1) were added to form an outer shell of biomolecules Then, calcium nitrate solution (0.5 mL), diammonium phosphate solution (0.5 mL) and CpG solution (0.2 mL, 63 µM) were subsequently added to form further layers of calcium phosphate and CpG. This led to triple-shell nanoparticles with the composition CaP/CpG + peptide/CaP/CpG (from core to shell). The final concentrations of CpG and GagL/gp70 peptide in the nanoparticle dispersion were 65.3 µg mL−1 (or 10.3 µM) and 20.4 µg mL−1, respectively. All inorganic salts were of p.a. (pro analysi) quality. The particles were characterized by scanning electron microscopy (SEM) and dynamic light scattering in the same way as described .
TLR-ligand, viral peptides and CCR4 antagonist
The phosphorothioate-modified class B CpG 1826 was purchased from Eurofins MWG Operon (Ebersberg, Germany). The FV protein derived Gag and gp70 peptide sequences containing MHC I and MHC II-restricted epitopes were synthesized with the following sequences: GagL85–93, CCLCLTVFL; gp70123–141, EPLTSLTPRCNTAWNRLKL (JPT Peptide Technologies GmbH, Berlin Germany). The CCR4 antagonist CAS 864289-85-0 was purchased from Merck Millipore (Darmstadt, Germany).
Antibodies and flow cytometry
The monoclonal antibodies αCD4 (clone RM4-5), αCD8 (clone 53–6.7) and αCD43 (clone 1B11) were obtained from BD Biosciences Pharmingen (Heidelberg, Germany). Monoclonal anti-human allophycocyanin (APC)-conjugated αGzmB antibody (clone GB12, Invitrogen, Karlsruhe, Germany) was used for intracellular granzyme B staining. The αFoxp3 antibody (clone FJK-16s) was purchased from ebioscience (Frankfurt, Germany). Intracellular staining with αFoxp3 and αGzmB was performed as described previously . To detect FV-specific CD8+ T cells a PE-conjugated recombinant MHC-class I H2-Db tetramer (Beckman Coulter) presenting the FV GagL85-93 peptide was used. Data was acquired by using an LSR II instrument using DIVA software (BD Biosciences).
Friend virus and chronic infection
A lactate dehydrogenase-elevating virus (LDV) containing B-cell-tropic, polycythemia-inducing Friend virus-complex (FV) was obtained from BALB/c mouse spleen cell homogenate 14 days post infection. To induce chronic FV infection, naïve FV-resistant C57BL/6 mice were infected with 15,000 spleen focus forming units (SFFU).
Vaccination of mice
For anti-viral therapeutic vaccination of chronically FV infected and vaccination of naïve mice for in vivo DC activation studies mice were immunized subcutaneously with 100 µL PBS or CaP nanoparticles functionalized with CpG/GagL/gp70 (10.3 µM CpG and 40.8 µg mL−1 GagL/gp70 peptide; CaP/CpG/GagL/gp70/CaP/CpG) in both hind footpads (50 µL each) 6 weeks post FV infection as described before .
Depletion of Foxp3+ regulatory T cells
To deplete Foxp3+ regulatory T cells, DEREG mice were injected intraperitoneally with diphtheria toxin (DT) (Merck, Germany) diluted in PBS. 800 ng in total per mouse was injected 4 and 2 days before as well as 2 and 4 days after vaccination with PBS or functionalized CaP NPs.
IFN-γ ELISpot assay
Interferon-γ-producing cells were evaluated by ELISpot with a mouse ELISPOTPLUS kit (MABTECH AB, Nacka Strand, Sweden). For evaluation of Interferon-γ-producing cells after therapeutic vaccination of chronically infected mice, spleen cells or lymph node cells from a cell pool of brachial, axillary, inguinal, and popliteal lymph nodes were restimulated. Restimulation of 2.5 × 105 cells was done with 5 µg mL−1 GagL85–93 or gp70123–141 peptide in a 96-well ELISPOT plate for 24 h. IFN-γ-spots were stained according to the manufacturer’s instructions. After the plate was dried, the number of spots was counted by an AID ELISPOT reader using ELISPOT 6.0 software (Mabtech, Nacka Strand, Sweden).
Infectious center assay
Determination of viral loads by infectious center assay was performed as described previously . Spleens were rinsed with RPMI containing 10 % FCS and 50 µg mL−1 penicillin/streptomycin. Spleen cells were counted and serially diluted before seeding them onto Mus dunni cells and incubated under standard tissue culture conditions for 3 days, fixed with ethanol and labeled with the primary F-MuLV Env-specific MAb 720. After washing, a secondary horseradish peroxidase (HRP)-conjugated rabbit antimouse Ig antibody (Dako, Hambrug, Germany) was added. Foci representing infectious centers were detectable after adding of aminoethylcarbazole (Sigma-Aldrich, Deisenhofen, Germany) as substrate for HRP. Foci were counted and infectious centers (IC)/spleen values were calculated.
Statistical analysis was performed by using student’s t test or One-way ANOVA to compare multiple groups using Bonferroni’s multiple comparison test. Data analysis was performed using Prism 6.0 software (GraphPad, La Jolla, CA). Statistical significance was set at the level of p < 0.05.
Animal experiments were performed in strict accordance with institutional, state, and federal guidelines. The protocol was approved by the North Rhine-Westphalia State Agency for Nature, Environment and Consumer Protection (LANUF) (Permit Number: G1433/14). All efforts were made to minimize suffering.
TK designed the experiments, performed the experiments, analyzed the data, did the statistical analysis and wrote the paper. OR, WB, VS and WH carried out experiments. ME, JB and UD designed experiments. TS provide DEREG mice and designed experiments. AMW conceived the experiments and wrote the paper. All authors read and approved the final manuscript.
We thank Patrick Juszczak, Mechthild Hemmler-Roloff and Svenja Groten for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB/Transregio 60 and GRK1949).
The authors declare that they have no competing interests.
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