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Preclinical evaluation of an mRNA HIV vaccine combining rationally selected antigenic sequences and adjuvant signals (HTI-TriMix)

Guardo, Alberto C.; Joe, Patrick Tjok; Miralles, Laia; Bargalló, Manel E.; Mothe, Beatriz; Krasniqi, Ahmet; Heirman, Carlo; García, Felipe; Thielemans, Kris; Brander, Christian; Aerts, Joeri L.; Plana, Montserrat on behalf of the iHIVARNA consortium

Author Information
doi: 10.1097/QAD.0000000000001276

Abstract

Erratum

The authors of these papers inform the readers that the study product iHIVARNA-01 had an error - the RNA sequence contained by mistake a second start codon in front of the HTI immunogen coding sequence.

This error is likely to influence the expression of the HTI protein from the mRNA vaccine. The degree to which this impacted the expression of HTI remains unclear. The results of the preclinical study showed an induction of an immunogen specific T-cell response in mice which could not be correlated with known HTI expression, as the expression level of the HTI protein had not been assessed. Similarly, in the clinical studies, the level of expression of the immunogen was not quantified.

The approval of the trial application for iHIVARNA-01 was based on the outcomes of in-vivo potency assays developed to analyse the functionality of the HIVACAT construct, and an in-vitro DC assay of the erroneous and correct HIVACAT/HTI mRNA.

The in-vivo assay showed a positive immune read out after intranodal immunization of mice with the HIVACAT (HTI) mRNA combined with murine TriMix. In addition, a comparison between the HIVACAT and HIVACAT-DCL construct – the latter with only one start codon and codifying for HIVACAT T immunogen - revealed no significant differences in the in-vivo potency assay.

The in-vitro DC assays tested the erroneous and correct HIVACAT/HTI mRNA. The correct HIVACAT/HTI mRNA also encoded a cytoplasmic region of human DC-LAMP sequence to improve the antigen presentation via MHC class II. Our preliminary results showed that both versions had quite similar effects on DC maturation and induced T-cell proliferation and Th1 cytokine at almost the same levels.

However, in summary as the level of expression of HTI was not measured it is unknown how the product error could have affected the results in this clinical study.

AIDS. 33(12):1957, October 1, 2019.

Introduction

Although it is clear that the immune system can play an important role in controlling HIV-1, HIV-1-specific T-cell frequencies wane with prolonged viral suppression, resulting in a weaker immune response against HIV replication [1]. Thus, boosting the immune system through therapeutic vaccination could offer an interesting alternative to lifelong cART. A variety of candidates have already been tested in nonhuman primates and some have also entered in human therapeutic trials [2]. However, despite serious effort, therapeutic vaccines against HIV-1 have shown modest efficacy at best [3]. Recently, however, some authors have provided evidence for a clinical benefit of immune-based interventions [4,5].

A major hurdle for vaccine formulations is the high mutational frequency of the virus leading to the emergence of escape variants [6,7]. To address this question, we have selected an immunogen based on the screening of three large cohorts of HIV-infected individuals from three different continents. The results of these studies yielded 16 regions in HIV-1 Gag, Pol, Vif, and Nef proteins that were preferentially targeted by individuals with low viral loads. These fragments were more conserved than the rest of the genome and elicited responses of higher functional avidity and broader variant cross-reactivity than responses to other regions [8,9].

In addition to the choice of antigen, the choice of immunostimulatory agents was shown to be important for the expansion and functionality of HIV-specific T cells [10]. Thus, we have previously shown that electroporation of human dendritic cells with TriMix, a compound mRNA formula encoding for CD40L, CD70, and a constitutively active Toll-like receptor 4 (caTLR4), resulted in a more mature phenotype and improved cytokine release [11]. These results were confirmed in a clinical setting where melanoma patients were vaccinated with dendritic cells electroporated with mRNA encoding TriMix and tumor antigens [12–14].

One of the main drawbacks of dendritic cell-based vaccines is that a personalized vaccine needs to be prepared for each patient, which is costly, cumbersome, and requires specific expertise and infrastructure. In this regard, intranodal mRNA vaccination could provide the potency of traditional dendritic cell vaccines at a reduced cost while circumventing their scalability issues [15,16]. Indeed, we demonstrated that when mRNA was delivered intranodally, it was almost exclusively taken up by CD11c+ dendritic cells, thereby underlining its potential to be used for in-vivo dendritic cell modification. Furthermore, a vaccine consisting of mRNA encoding a combination of TriMix and tumor antigens resulted in a prolonged survival in tumor-bearing mice upon intranodal vaccination [17].

In summary, although HIV-specific immune responses are clearly enhanced after vaccination with dendritic cells [18–20], the clinical responses induced by dendritic cell-based vaccines are generally disappointing [15,16,21]. In this manuscript, we describe the preclinical evaluation of an mRNA-based therapeutic HIV-1 vaccine consisting of TriMix and HIVACAT T-cell immunogen (HTI) in a human setting in vitro and in vivo in mice.

Material and methods

Sample donors and animals

A total of 13 HIV-1-infected patients were recruited in a single center (Hospital Clinic, Barcelona, Spain) according to the following inclusion criteria: asymptomatic chronic HIV-infected patients with undetectable viral load (<37 copies/ml), stable cART treatment for at least 6 months, and baseline CD4+ T-lymphocytes above 450 cells/μl. All the individuals gave informed written consent and this study was reviewed and approved by the Institutional Ethical Committee board of Hospital Clinic (Barcelona, Spain).

For mouse experiments, female 6-week-old to 12-week-old C57BL/6 mice were purchased from Charles River (Wilmington, Massachusetts, USA) and handled according to the guidelines and regulations of the Animal Care Committee of the Vrije Universiteit Brussel.

Generation of monocyte-derived dendritic cells

Peripheral blood mononuclear cells (PBMCs) were isolated from a 150-ml sample of EDTA-treated venous blood within 1 h after the blood sampling by means of standard Ficoll gradient centrifugation. Monocytes were obtained as described elsewhere [5]. Briefly, PBMC were incubated for 2 h at 37°C in 5% CO2 with dendritic cell medium, consisting of serum-free XVIVO-15 medium (Bio-Whittaker, Walkersville, Maryland, USA) supplemented with 1% autologous serum, 50 μg/ml gentamycin (Braun B. Melsungen, Germany), 2.5 μg/ml fungizone (Bristol-Myers Squibb, Munchen, Germany), and 1 μmol/l zidovudine (Genericos Españoles Laboratorios, Las Rozas, Spain) to avoid potential replication of endogenous HIV-1. After 2 h of incubation, monocytes were confirmed to be in monolayer and nonadherent cells were removed by three washes with warm (37°C) phosphate-buffered saline (PBS). Subsequently, monocytes were cultured for 5 days in dendritic cell medium supplemented at days 0 and 3 with human recombinant IL-4 and granulocyte macrophage colony stimulating factor (GM-CSF) (1000 IU/ml, each, Cellgenix, Freiberg, Germany). After 6 days of culture, immature dendritic cells (iDCs) were obtained after several washes with cold and sterile PBS.

In-vitro transcription of mRNA

The cDNA sequences (GeneArt; Thermo Fisher Scientific, Regensberg, Germany) encoding for CD40L, CD70, caTLR4, and HTI were codon optimized for human or mouse (CD40L and CD70) use. The cDNA sequence was then cloned in the pEtherna vector (eTheRNA, Brussels, Belgium) for enhanced stability and translation of the mRNA.

Plasmids were linearized and in-vitro transcribed using a T7 polymerase runoff reaction. mRNA constructs were capped using antireverse cap analog and the poly (A) tail was transcribed from the plasmid sequence. The mRNA was then resuspended in endotoxin-free water or 0.8 Ringer lactate (Baxter, Melbourne, Australia) for in-vitro or in-vivo experiments, respectively.

Intranodal injection

Intranodal injections were performed by surgically exposing the inguinal lymph node of anesthetized animals following injection of 12.5 μg TriMix, 37.5 μg HTI, or 37.5 μg HTI + 12.5 μg TriMix mRNA, and closing of the wound. For multiple immunizations, injections were alternated between the left and right inguinal lymph nodes and given every 2 weeks.

Cell transfections

Prior to mRNA exposure, iDCs were washed twice with serum-free Iscove's Modified Dulbecos Medium and resuspended to a final density of up to 4 × 106 cells/ml in Opti-MEM (Gibco BRL Alcobendas, Spain). The following electroporation conditions were tested: HTI alone, TriMix alone, HTI + TriMix, and control mRNA (Stagment eGFP-mRNA, Miltenyi, Cologne, Germany). For each condition, the cell suspension was mixed with the appropriate amount of mRNA [20 μg of HTI and/or 20 μg of TriMix (caTLR4 + CD40L + CD70)], in a final volume of 0.2 ml of Opti-MEM and were electroporated in a 0.4-cm cuvette using a Gene Pulser II electroporation system (Biorad, Hercules, California, USA). Electroporation conditions were as follows: voltage of 300 V, capacitance of 150 μF, resistance of 800 Ω resulting in a pulse time of 9–10 ms. After electroporation, Iscove's Modified Dulbecos Medium + 10% human AB serum was added to the cell suspension and incubated for 2 h at 37°C in 5% CO2, before use. An additional condition was included, whereby HTI + TriMix mRNA was passively pulsed on dendritic cells. In this case, the cells were incubated in a final volume of 200 μl for 15 min at 37°C. A maturation cocktail (IL1β, TNFα, IL-6, and prostaglandin E2) was used as a control for the maturation profile determinations [22].

Proliferation assay

iDCs were harvested on day 7 and fresh autologous monocyte-depleted PBMCs were obtained from a second blood extraction. Briefly, cells were stained with 5 μmol/l carboxyfluorescein succinimidyl ester (CFSE) using the CellTrace CFSE proliferation kit (Invitrogen, Carlsbad, California, USA) in a 10-ml conical tube [23]. T-cell proliferation was measured by flow cytometric analysis of CFSE dilution after 7 days of coculture.

Cytokine and chemokine secretion measurement

Modified dendritic cells and autologous monocyte-depleted PBMCs were cocultured as described above, and the supernatants were collected after 48 h and 6 days, respectively, to be analyzed by Multiplex Luminex assays (Cytokine Human 25-Plex Panel; Invitrogen, Carlsbad, California, USA) following the manufacturer's protocol.

Flow cytometry

The immune phenotype upon dendritic cell maturation was assessed for each condition by a four-color panel using the following mAbs (BD Biosciences, San Jose, California, USA): CD3 and CD40 fluorescein isothiocyanate (FITC), CD80, CD83, CD86, CCR7 and CD169 (Siglec-I)-phycoerythrin, CD14-allophycocyanin (APC), and human leukocyte antigen - antigen D related (HLA-DR) peridinin chlorophyll protein (PerCP). Relevant mouse immunoglobulin isotypes conjugated with PerCP, phycoerythrin, FITC, or APC were always used as controls for nonspecific binding.

For the analysis of resident cell subsets in lymph nodes exposed to different mRNA conditions, the following mAbs were used: PercP-conjugated anti-CD11c, FITC-conjugated anti-CD11b and Pacific Blue-conjugated anti-CD14, APC-conjugated anti-CD83 (all from BD Pharmingen, San Jose, California, USA), Alexa Fluor-700-conjugated anti-CD3 (BioLegend, San Diego, California, USA), BV605-conjugated anti-CD4 (Life Technologies, Eugene, Oregon, USA), APC-Cy7-conjugated anti-CD19, phycoerythrin-conjugated anti-HLA-DR (BD Pharmingen), and Live/dead Fixable Blue Dead Cell Stain Kit (Life Technologies).

Samples were acquired using a FACSCalibur or LSR Fortessa flow cytometers (BD Biosciences) and analyzed using FlowJo Software (Tree Star, Ashland, Oregon, USA).

ELISPOT assay for IFN-γ release

Ex-vivo IFN-γ production by T cells was measured by ELISPOT as previously described [24]. In a first instance, an ELISPOT was performed using fresh autologous lymphocytes stimulated by different dendritic cell conditions: alone Roswell Park Memorial Institute medium/10% fetal calf serum negative control, with differentially modified dendritic cells and with phytohemagglutinin (5 μg/ml; Sigma, St Louis, Missouri, USA) as a positive control. In a second setting, lymphocytes that were cultured the week before with differentially modified dendritic cells were used alone, as negative controls, or in the presence of different HIV peptides pools at a final concentration of 2 μg/ml (IN and OUT of the sequence of the insert; Fig. 2). Again, phytohemagglutinin was included as a positive control [25]. In both cases, the cells were lysed after 16–18 h by washing six times with PBS/0.05% Tween 20, and the wells were incubated for 3 h with 1 μg/ml of biotin-labelled, anti-IFN-γ mAb 7-B6-1 (Mabtech, Stockholm, Sweden). To visualize the spot forming foci (IFN-γ-secreting cells), the wells were washed six times with PBS Tween and treated for 1 h with streptavidin-alkaline phosphatase (Mabtech). After this incubation, the wells were washed again and 100 μl/well of chromogenic alkaline phosphatase-conjugated substrate (BioRad) was added.

Fig. 2
Fig. 2:
In-vivo cytotoxic T-cell responses against peptide-loaded target cells in mice after prime-boost vaccination with HTI and TriMix mRNA.CellTraceViole-labeled target cells loaded with HTI peptides were administered 5 days after prime vaccination or 4 days after additional boost vaccines in C57BL/6 mice. Lymph nodes and spleens were isolated the day after and specific lysis was analyzed by flow cytometry. Each symbol represents one mouse. Data are depicted as median + interquartile range and summarize the results of four independent experiments with eight to 16 mice per group.

For murine samples, splenocytes were stimulated for 36 h with HTI peptide pools at a total concentration of 14 μg/ml per peptide. IFN-γ detection was performed according to the murine IFN-γ kit from Diaclone (Stamford, Connecticut, USA). Spot-forming cells (SFC) were counted using an AID ELISPOT reader (Autoimmun Diagnostika GmbH, Germany).

Results were considered positive if the number of SFC/106 cells in stimulated wells was two-fold higher than that in unstimulated control wells, and if there were at least 50 (or 20 in murine samples) SFC/106 cells after background subtraction.

Lymph node assay

Inguinal or axillary human lymph nodes were obtained from deceased anonymous donors kindly provided by Dr J. Martorell and Dr Rull from Hospital Clinic and were processed as indicated elsewhere [26]. Briefly, we dissected tissue into blocks of uniform size and transferred them to a 96-well round bottom plate. Lymph node fragments were exposed to the following conditions: negative control with Opti-MEM only, truncated nerve growth factor receptor) mRNA control (40 μg) [27], and HTI + TriMix (20 μg+20 μg) mRNAs. After 4 and 24 h, supernatants were collected and the cells were harvested, to perform Luminex assays and flow cytometry, respectively, as described above.

In-vivo cytotoxicity assay

Splenocytes from C57BL/6 mice were pulsed for 1 h 30 min with 0.5 μg/ml of HTI peptides. Cells were then labelled with 1.5 μmol/l of CellTraceViolet (Life Technologies) according to the manufacturer's protocol. Peptide-pulsed cells were then mixed with equal amounts of nonpulsed splenocytes labelled with 0.15 μmol/l CellTraceViolet and injected intravenously. Lysis of target cells was analyzed by flow cytometry 18 h later. Specific lysis was calculated as described [28].

Statistical analysis

Data analysis and comparisons for the different parameters were made using parametric (Student's t-test) and nonparametric (Mann–Whitney or Wilcoxon signed rank test) tests as appropriate. For comparisons of multiple groups the Kruskal–Wallis test was performed with correction using Dunn's multiple comparison test. Statistical analysis was performed using SPSS 18.0 (Windows) software (SPSS Inc., Chicago, Illinois, USA) and GraphPad Software (GraphPad Prism version 5.00; La Jolla, California, USA). For all analyses, the level of significance was set at P < 0.05.

Results

Immature dendritic cells electroporated with HTI and/or TriMix mRNA induce potent antigen-specific T-cell responses in vitro

iDCs obtained from asymptomatic HIV-infected individuals were electroporated with different mRNA combinations. The expression of different costimulatory molecules (CD83, CD80, CD86, and CCR7) was then measured to determine the maturation profile of these cells. As expected, dendritic cells electroporated with TriMix + HTI mRNA showed a marked increase in the expression of these surface molecules (Fig. 1a), especially when TriMix mRNA was present. In fact, TriMix alone obtained a better maturation profile than HTI alone (data not shown). In conclusion, all the maturation markers evaluated improved their expression after TriMix (+HTI) electroporation, although not at the same level as the conventional maturation cocktail did. On the other hand, passive diffusion of both components was inefficacious except for the slight increase of CD83 and CCR7 detected.

Fig. 1
Fig. 1:
Phenotypical and functional characterization of electroporated human dendritic cells in coculture with autologous monocyte-depleted PBMC.(a) We analyzed the maturation profile of these cells by flow cytometry with the indicated mAbs. (b) Proliferation of CD4+ and CD8+ T cells from HIV-infected patients upon stimulation with HTI + TriMix-loaded dendritic cells (T cell : dendritic cell ratio of 7 : 1). (c) Cytokine and chemokine production by Luminex assay. To characterize the effects of HTI + TriMix, the levels of secreted cytokines and chemokines in the culture supernatants of cocultures between dendritic cells and PBMC, were assessed using the Luminex technology (25 multiplex). Wilcoxon rank test was performed; for all analyses, the level of significance was set at P < 0.05. EP, electroporation; SEA, Staphylococcus aureus enterotoxin A.

We also evaluated the T-cell stimulatory capacity of these human-electroporated dendritic cells measuring proliferation and cytokine production of autologous monocyte-depleted PBMCs in coculture with modified dendritic cells. As was previously reported [29], proliferation of both CD4+ and CD8+ lineages was significantly increased after TriMix electroporation (alone or in combination with HTI) and to a lesser extent after HTI electroporation alone (Fig. 1b), whereas passive diffusion yielded the poorest response. As expected from the design of the HTI immunogen, proliferation was higher in the CD8+ T-cell lineage than in the CD4+ population after HTI + TriMix stimulation.

Similar observations were made regarding the cytokine secretion profile (Fig. 1c) where several cytokines were significantly increased after TriMix stimulation. In this context, only IFN-γ was significantly increased upon HTI + TriMix stimulation compared with either HTI or TriMix alone. Overall, 19 out of 25 cytokines were within the normal range (Supplementary table, http://links.lww.com/QAD/A991).

Intranodal immunization with HTI + TriMix mRNA induces potent cytotoxic T-cell responses in mice

To evaluate whether we could extrapolate our findings in vivo, we performed multiple intranodal immunizations in C57BL/6 mice with the indicated formulations. A single immunization with HTI + TriMix induced antigen-specific cytotoxic T-lymphocyte (CTL) response in the lymph nodes. These responses were significantly increased in both lymph nodes and spleen, when a second vaccine was given (Fig. 2). The administration of a third vaccine did not further improve CTL activity in the lymph nodes of HTI + TriMix-vaccinated animals and only slightly in the HTI group. Cytotoxic responses were however substantially increased in the spleens of both HTI and HTI + TriMix groups with a trend toward higher CTL responses in the former. This suggests that although the inclusion of TriMix to the HTI vaccine formulation enhances CTL responses after one and two immunizations, its effect diminishes after three vaccinations. Together, these data demonstrate that in mice, intranodal delivery of HTI or HTI + TriMix mRNA induces systemic antigen-specific CTL responses.

HTI antigen presence is critical to enhance IFN-γ production

Next, we assessed whether the results obtained by Luminex assays could be quantified by ELISPOT at different times. We observed that IFN-γ production at day 1 was significantly increased in the presence of the HTI immunogen, whereas TriMix alone was not able to induce a relevant response (Fig. 3a). After 6 days of stimulation with the indicated HIV peptide pools, IFN-γ production was increased in all the cases (Fig. 3b) in comparison with the basal condition (no pools added). Finally, and with the purpose to test if the response against HIV was driven by the sequence included in the HTI, the elicited response against a pool of Gag peptides (included in the sequence of the insert) and a pool of Pol peptides (not present in the immunogen sequence) was performed. As shown in Fig. 3c, we detected significant differences between responses to these pools when the HTI was present.

Fig. 3
Fig. 3:
ELISPOT assay: IFN-γ release by autologous monocyte-depleted PBMCs in the presence of electroporated dendritic cells at different times.(a) Representative example of ELISPOT plate at day 1 from one HIV patient after overnight stimulation with HTI and/or TriMix-loaded dendritic cells and its respective quantification (n=8). (b) ELISPOT evaluated after 6 days of culture in the presence of the indicated Gag (p24) and Pol peptide pools (n=8). (c) Comparison of ELISPOT at day 6 between different conditions (electroporated mRNA and passive diffusion) after stimulation with Gag IN and Pol OUT peptide pools (n=4). For all analyses, the level of significance was set at P < 0.05.

Immunization with HTI + TriMix induces IFN-γ responses against multiple regions of the HTI sequence in mice

To further extend our study on the immune responses generated in vivo, we evaluated the targeted regions of the HTI sequence in mice after triple immunization.

Both HTI and HTI + TriMix mRNA-vaccinated animals targeted multiple regions of the HTI sequence in IFN-γ ELISPOT (Fig. 4a). Consistent with the in-vivo CTL responses, we did not observe a difference in the breadth and strength of the response against HTI between the two groups after triple vaccination (Fig. 4b).

Fig. 4
Fig. 4:
IFN-γ ELISPOT analysis of targeted HTI regions by immune responses induced by the vaccine in mice.Splenocytes from immunized C57BL/6 mice were isolated 5 days after the third immunization and restimulated with 17 different peptide pools covering the HTI immunogen. Results are shown from three independent experiments with nine mice per group. (a) Summary of the breadth of the response displayed as the number of peptide pools targeted per mouse. (b) Overall strength of the response against the HTI immunogen calculated as the total sum of the IFN-γ responses against all the peptide pools. Results from (a) and (b) are depicted as median ± IQR. (c) Strength of the IFN-γ responses against the individual peptide pools (left graph) which are summarized according to their region (right graph). Results are depicted as mean ± SEM.

Both vaccinated groups showed similar patterns in targeting the HTI sequence, with the highest responses being detected against Gag p17, Gag p24, integrase, and Vif regions (Fig. 4c). Interestingly, a small number of mice vaccinated with HTI + TriMix mRNA also elicited responses against the Nef region, which was not observed in the group vaccinated with only HTI mRNA.

All these data demonstrate that triple immunization with HTI or HTI + TriMix mRNA induces broad immune responses against multiple regions of the HTI construct.

Lymph node explants exposed to HTI + TriMix mRNAs show that resident dendritic cells improved their maturation profile and modified the cytokine milieu

Our previous findings with iDCs prompted us to analyze, whether codelivery of TriMix and HTI could promote a T-cell-attracting and activating environment in a more physiological scenario. In this context, we evaluated the expression levels of maturation markers after mRNA exposition using human lymph node explants. We compared three different conditions: basal (no mRNA addition), truncated nerve growth factor receptor control mRNA, and HTI + TriMix mRNA. Our results suggest that exposure to any mRNA contributed to dendritic cell maturation, although HTI + TriMix was the most efficient in this respect (Fig. 5a). In fact, we have observed that CD11c+ cells were overrepresented after HTI + TriMix mRNA exposition (Fig. 5b) and that this dendritic cell population could be responsible for the mRNA uptake, as was described in previous work [17]. Finally, we measured the cytokine and chemokine profile 4 or 24 h after mRNA exposure. We observed that both IFN-γ and IFN-γ-inducible protein 10 (Fig. 5c) were significantly increased after 24 h of HTI + TriMix exposition. Moreover, proinflammatory cytokines such as IL-1β were also increased, whereas inflammation antagonists such as IL-1RA showed an opposite tendency. The elevated levels of chemokine (C-X-C motif) ligand 8 (CXCL8) and GM-CSF, respectively, could be responsible for the recruitment and maturation of important cell populations such as monocytes, macrophages, or neutrophils (Fig. 5c).

Fig. 5
Fig. 5:
Flow cytometry analysis of lymph node explants exposed to different mRNAs conditions (basal, tNGFR, and HTI + TriMix mRNA).(a) Maturation profile (CD80, CD83, CD86, CCR7, and Siglec-I) obtained from a dendritic cell subset isolated from a lymph node explant. (b) Gating criteria used to identify isolated cells from a lymph node explant. (c) Example of six different cytokines detected by Luminex in the supernatants from lymph node explants cultures at two different time points (4 and 24 h; n=5). Wilcoxon rank test was performed; for all analyses, the level of significance was set at P < 0.05. tNGFR, truncated nerve growth factor receptor.

Discussion

Here, we present the results of both in-vitro and in-vivo assays carried out with a new mRNA vaccine strategy that comprises two different components: an HIV immunogen (HTI) and an adjuvant (CD40L, CD70, and caTLR4: TriMix). Overall, we have observed that the combination of HTI + TriMix can induce a potent immune response based on the following findings. First, the maturation profile of human dendritic cells was enhanced after mRNA electroporation. This finding, observed previously in cancer research [17,30], was maintained within the HIV context. Regarding IFN-γ production, our results showed that the presence of HTI, independently of TriMix, generates an earlier and specific response observed even after overnight incubation. Later on, this response was potentiated by the adjuvant and became even higher than in the case of HTI alone, suggesting that the initial specific response could be significantly improved by the milieu created by the adjuvant [17]. Another clue about the specificity of the elicited response could be found by the fact that the Gag peptide pool used inside the insert sequence showed a higher response than the peptides outside the HTI sequence. Similar results were obtained previously by Van Gulck et al.[31] where dendritic cells loaded with consensus or autologous Gag mRNA, were able to expand HIV-specific T-cell responses in vitro[32]. In regard to the proliferation rate determined by CFSE dilution, we observed an important increase in both lineages after incubation with HTI + TriMix-modified dendritic cells, especially for CD8+ T cells. This finding is not surprising as the design of the sequence of the insert was specifically created as a broadly applicable T-cell immunogen restricted by a wide array of HLA class I alleles [8]. These T-cell responses to specific HIV-1 proteins and protein subunits have been associated before with relatively superior viral control in vivo[33]. Their promotion constituted a main goal of an effective HIV immunogen because it has been shown that T-cell frequencies decline with prolonged viral suppression [1,34]. Our results suggest that, at least in vitro, iDCs derived from HIV-1-infected patients under full cART suppression and electroporated with mRNA can induce CD4+ and CD8+ T-cell proliferation.

We also observed significant levels of cytokine production by T cells upon stimulation with electroporated dendritic cells. In this regard, a study by Pollard et al.[35] reporting on the feasibility of using mRNA encoding the HIV Gag antigen complexed with DOTAP/DOPE (1,2-dioleoyl-3-trimethylammoniumpropane/dioleoylphosphatidylethanolamine) as an immunization strategy shed some light on this issue. As in our case, they observed a potent IFN-γ production when T cells were stimulated with dendritic cells that were exposed to their formulation. In our hands, significantly increased levels of proliferative (e.g. IL-2), inflammatory (e.g., TNF-α or IL-6) cytokines as well as chemokines (CXCL8, CCL3) were observed, especially when TriMix was present (alone or in combination with HTI).

Intranodal vaccination with dendritic cells has been proposed as a more efficient alternative to the more conventional subcutaneous and intradermal routes of administration. Although considered more feasible, recent results published by Gandhi et al.[21] showed that intradermal immunization with HIV-1 Gag and Nef-transfected dendritic cells did not induce significant IFN-γ responses. In the context of ‘naked’ mRNA vaccination, intranodal delivery results in both higher protein expression and protective antigen-specific T-cell responses [36] compared with subcutaneous and intradermal injection.

To test passive diffusion of mRNA into lymph node-resident dendritic cells in a more physiological way, we analyzed the effect of mRNA exposition in a human lymph node explant model. Our results on maturation markers of dendritic cells and cytokine production revealed that both were promoted, especially upon HTI + TriMix exposure. These results confirm that mRNA addition enhances the stimulatory capacity of lymph node-resident dendritic cells, as was previously observed for the dendritic cell electroporation assays. However, it is not clear whether the intrinsic adjuvant effect of mRNA is sufficient to fully exploit the immunostimulatory capacity of dendritic cells or whether additional stimulation signals are required [37]. Although important differences still exist with what happens in a lymph node in vivo, the lymph node explant model was a good approximation for testing mRNA processing and expression by resident iDCs [38].

To further assess the feasibility of intranodal mRNA delivery for vaccination purposes, we demonstrate that intranodal injection of HTI + TriMix mRNA induces broad T-cell responses capable of antigen-specific cell lysis in mice. In parallel with the human in vitro data, we found that the inclusion of TriMix mRNA enhanced CTL responses after one and two immunizations compared with HTI alone, especially in the lymph nodes of vaccinated animals. However, although three immunizations were necessary for robust systemic responses, the addition of TriMix did not result in a higher CTL activity in this case. This finding was further confirmed by IFN-γ ELISPOT analysis in which the immune responses induced by HTI and HTI + TriMix resulted in broad responses of equal strength against multiple but similar regions after three immunizations.

The observation that TriMix-HTI did not further enhance T-cell responses compared with HTI alone after three immunizations could be explained by several possible mechanisms. Explicitly, mRNA can activate inflammatory pathways upon binding to TLR3, 7, and 8 [39–42]. The potency of the intrinsic adjuvant properties of mRNA was recently demonstrated in a study in which repeated intratumoral injections of high amounts of ‘irrelevant’ control mRNA delayed tumor growth in vivo[43]. Therefore, it is possible that mRNA by itself is sufficient to act as a stimulator after three immunizations, thereby overshadowing the additional effects of TriMix. Another possible explanation can be found in the controversial effects of the CD70 molecule included in the TriMix. Although the binding of CD70 to its receptor CD27 is crucial for T-cell responses, chronic stimulation of this pathway has been shown to drive T-cell exhaustion [44–46]. Therefore, although TriMix improves immune responses after two vaccinations, further injections might overactivate the CD27/CD70 pathway causing it to counteract the stimulatory effects of TriMix.

Our study has a number of limitations that should be noted. First, the potent effect observed after TriMix addition suggests that TriMix alone could promote a context of inflammation or activation, which is not specifically directed against HIV. Regarding ELISPOT assays, we cannot discard the participation of innate immune cells (e.g., natural killer or natural killer T cells) because they were not depleted from the samples. Further and related with the evaluation of maturation markers, we observed that CD169/Siglec-1, which is involved in transinfection of HIV, [47] was also upregulated. In any case, we hope to answer questions related with safety and efficacy in the near future when an ongoing phase I clinical trial (approved by the Spanish regulatory authorities; Spanish Agency of Medicines and Medical Devices), whereby HIV-1-infected individuals on cART receive different doses of TriMix alone or in combination with HTI mRNA, will be completed.

In summary, our results suggest that the selected combination is an effective option on the basis of complementarity of its mode of action with that of the vaccine format it will be combined with [48]. In that regard, the efficacy of mRNA administered into lymph nodes depends on its uptake and its ability to create a CTL-inducing milieu. These findings may pave the way for therapeutic HIV vaccine strategies based on naked antigen-encoding RNA.

Acknowledgements

We are grateful to Dr J. Martorell and Dr Rull from Hospital Clinic for kindly providing us with lymph node explants from deceased anonymous donors. We would also like to thank Špela Jug for expert assistance with the animal experiments.

The work was partially supported by FP7-HEALTH-2013-INNOVATION-1, 602570-2, HIVACAT, RIS, and Wetenschappelijk Fonds Willy Gepts of the UZ Brussel to J.L.A. P.T.J. was funded by a PhD grant from the Flemish agency for innovation by science and technology (IWT); FIS PI15/00480, from Plan Nacional de I+D+I and cofinanced by ISCIII-Subdirección General de Evaluación and Fondo Europeo de Desarrollo Regional (FEDER).

Conceived and designed the experiments: A.C.G., P.T.J., B.M., C.B., J.L.A., K.T., F.G., and M.P. Performed the experiments: A.C.G., P.T.J., L.M., A.K., and M.E.B. Analyzed the data: A.C.G., P.T.J., J.L.A., and M.P. Contributed reagents/materials/analysis tools: C.H. and K.T. Wrote the article: A.C.G., P.T.K., J.L.A., and M.P.

Members of the IHIVARNA consortium (www.ihivarna.org)

Institut d’ investigacions Biomèdiques August Pi I Sunyer (IDIBAPS); Barcelona, Spain: Dr Felipe Garcia, Dr José M. Gatell, Dr J.A. Arnaiz, M.P., Dr Lorna Leal, A.C.G., and Maria José Maleno; Instituut voor Tropische Geneeskunde (ITM), Antwerp, Belgium: Dr Guido Vanham, Dr Eric Florence, Dr Pieter Pannus, Dr Jozefien Buyzea, and Leo Heyndrickx; Vrije Universiteit Brussels (VUB), Brussels, Belgium: K.T., J.L.A., Dr Sabine Allard, and P.T.; Institut de Recerca de la Sida (Irsi Caixa); Badalona, Spain: C.B., B.M., Dr J. Martinez-Picado, Dr Alex Olvera, Dr Miriam Rosas, Dr Maria Salgado, Marta Marszalek, and Sara Moron; Erasmus Universitair Medisch Centrum Rotterdam (EMC), Rotterdam, Netherlands: Dr Rob Gruters, Dr Marion Koopmans, Dr Wesley de Jong, Patrick Boers, Rachel Scheuer; eTheRNA, Brussels, Belgium: Dr Carlo Heirman, Dr Sonja Van Meirvenne; ASPHALION, Barcelona, Spain: Anna Graupera; and SYNAPSE, Barcelona, Spain: Ángel Honrado.

Conflicts of interest

There are no conflicts of interest.

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* Alberto C. Guardo, Patrick Tjok Joe, Joeri L. Aerts, and Montserrat Plana contributed equally to the writing of this article.

Keywords:

HIV vaccines; HTI; mRNA electroporation

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