The Combination of an mRNA Immunogen, a TLR7 Agonist and a PD1 Blocking Agent Enhances In-Vitro HIV T-Cell Immune Responses

The development of new strategies to achieve a functional cure for HIV remains a priority. We tested a novel HIV therapeutic vaccine using unmodified mRNA (TMEP-B) and mRNA modified by 1-methyl-3′-pseudouridylyl (TMEP-Bmod) expressing both a multiepitopic sequences from Gag, Pol, and Nef proteins, including different CD4 and CD8 T-cell epitopes functionally associated with HIV control in transfected monocyte-derived dendritic cells (MDDCs) obtained from HIV infected patients. In vitro assays were used to test the mRNAs alone and in combination with immunomodulator agents, such as the TLR-7 agonist Vesatolimod and the PD-1 antagonist Nivolumab to try to improve HIV-specific cellular immune responses. Combining the mRNAs with the immunomodulators enhanced HIV-specific T-cell responses, together with the secretion of IFNγ, IP10, MIP-1α, and MIP-1β, which are fundamental mediators of viral control. Our data suggest that the mRNA vaccine prototypes TMEP-B and TMEP-Bmod, when combined with Vesatolimod and/or Nivolumab, could achieve functional cure for patients with HIV.


Introduction
Antiretroviral therapy (ART) has significantly improved the morbidity and mortality associated with HIV [1]. However, it is unable to achieve cure or eradication. Thus, achieving functional cure remains the best approach, with promising results from several therapeutic vaccines. However, none has fully succeeded in controlling the viral replication of HIV.
Functional cure of HIV necessitates that a treatment induces not only an efficient immune response but also impacts the latent viral reservoir [2][3][4][5]. Nucleic acid (DNA/RNA) vaccines offer a novel and alternative strategy to conventional vaccines by directing the immune response against specific viral epitopes. Recently, our research group described the design and immunogenicity profile, in animal models, of a T-cell-based HIV-1 immunogen containing different domains of HIV-1 Gag, Pol, and Nef proteins expressed by DNA or modified vaccinia virus Ankara (MVA) vectors. This was named T-cell multiepitopic peptide (TMEP-B) [6,7]. Although DNA-based vaccines against HIV have been used in clinical trials, they have some clear limitations, such as the need for high doses when administered directly into the muscle, the need for electric devices to accelerate nucleic acid penetration into cells, and from having poor immunogenicity [8,9].
The direct in vitro administration of mRNA-based vaccine candidates into cells can lead to protein expression, providing new hope for a functional HIV cure [10,11]. These mRNA vaccines have many advantages with respect to efficient antigen expression efficiency. However, mRNA is fundamentally unstable, and the sequences and secondary structures that form (e.g., double helix, stem-loops structures) can be recognized by several innate immune receptors that could inhibit protein translation. Several methods with therapeutic relevance are being employed to improve the efficiency of translation, such as substituting modified nucleosides in the mRNA to make it non-inflammatory by avoiding type 1 IFN stimulation and allowing effective translation. It is also important for protein expression to achieve efficient intracellular delivery of mRNA in the cytosol, especially for mRNA administered systemically, to avoid its degradation and allow better cellular uptake and release. For this, lipid nanoparticles have been used successfully for mRNA release, even allowing for combinations of different mRNAs in a same formulation and improving the vaccine's effectiveness [11][12][13][14][15][16][17][18].
The latent viral reservoir is a major obstacle to achieving functional cure of HIV. To overcome this drawback, researchers have proposed combining a therapeutic vaccine with latency reversal agents (LRAs) [19]. In this way, the combination of mRNAs encoding for HIV proteins with different immunomodulatory agents would result in an interesting strategy that could be developed to achieve functional cure. In non-human primates, using a TLR7 agonist in combination with other immunotherapies induced a state of remission and perhaps even the complete cure of HIV infection [20]. Moreover, using a TLR7 agonist together with HIV-1 peptides in vitro led to increased cell degranulation, cytokine production, and cytotoxic activity [21]. Agent inhibitors of the PD1/PDL1 pathway are other possible candidates, given that the expression of PD1 in infected cells contributes to the establishment and maintenance of HIV latency [22]. Furthermore, PD-1 blockade with a monoclonal antibody or the interference between PD1 and PDL1 during antigenic presentation can increase HIV-specific T-cells and enhance HIV latency reversal in patients who have achieve suppression with ART [22][23][24][25].
Using an in vitro model with cells from HIV infected patients, we evaluated the potential of unmodified or modified mRNA TMEP-B that coded for a multiepitope protein, either alone or in combination with a TLR7 agonist (Vesatolimod) or a PD1 antagonist (Nivolumab), to induce efficacious HIV-specific T-cell immune responses.

Study Individuals
This study included patients with chronic stable HIV infection and receiving ART with follow-up at the Clinic Hospital in Barcelona, Spain. Patients were eligible if they consented to donating blood and had a plasma viral load (pVL) of ≤50 copies/mL and a CD4+ T-cell count of >400/mm 3 . All patients signed an informed consent form agreeing to the use of their clinical data for research purposes. This study was reviewed and approved by the institutional Ethics review board (HCB/2018/0305).

Study Samples
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque densitygradient centrifugation at 2000 rpm for 20 min at room temperature. The collected PBMCs were then washed twice with Dulbecco's phosphate-buffered saline and manually counted for viability using a trypan blue exclusion dye. Freshly obtained PBMCs were used to generate monocyte-derived dendritic cells (MDDCs).

MDDC's Generation
In order to obtain human monocytes, PBMCs were cultured in a 75 cm 2 cell culture flask for 2 h at 37 • C in a 5% CO 2 environment at a concentration of 3-4 × 10 6 cells/mL in X-VIVO 15 media (Lonza, MD, USA) supplemented with 1% inactivated autologous serum, gentamicin (50 µg/mL, B/Braun Medical, Melsungen, Germany), fungizone (2.5 µg/mL, Life Technologies, California, USA), and Zidovudine (1µM, Genéricos Españoles Laboratorios, Madrid, Spain). Monocytes were confirmed to be in a monolayer after incubation for 2 h, and the nonadherent cells (MNC) were removed by three washes with phosphatebuffered saline (PBSx1) ( Figure S1). Subsequently, monocytes were cultured for 6 days with 1000 U/mL each of recombinant human IL-4 (ProSpec, Rehovot, Israel) and recombinant human GM-CSF (ProSpec) on days 0 and 2. After 6 days, immature dendritic cells (iDCs) were obtained by adding cold PBSx1 to the 75 cm 2 cell culture flask to the harvest them by firmly tapping the flasks. This dislodged the loosely adherent dendritic cell (DC) clusters. MDDCs were harvested into a 50 mL conical tube and counted manually for viability using the trypan blue exclusion dye. Lymphocytes were collected from MNC fractions and frozen as PBMCs for use in mixed lymphocyte response assays across different DC preparations.

Flow Cytometry
The phenotypes of MDDCs, iDCs, and mature DCs (mDCs), together with the expression of markers related to the migration ability of DCs and T-cell proliferation, were assessed by flow cytometry using different combinations of mAbs ( Figure S2). The corresponding isotypes were used as controls. The viability and mortality of all cell populations were assessed using an Annexin V-PE/7-AAD Apoptosis Kit (Becton Dickinson) or the LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (ThermoFisher Scientific, Massachusetts, USA). Samples were acquired by FACSCanto II (BD Biosciences; San Jose, CA, USA) and analyzed with FlowJo (Tree Star, Ashland, OR, USA).

Transfection: Electroporation of MDDCs with Unmodified/Modified mRNA TMEP-B
The TMEP-B protein sequence was translated into an RNA/codon-optimized nucleotide sequence avoiding all the time the RNA inhibition and instability elements, as well as intended splicing sites. The synthetic TMEP-B gene (1868 bp) was assembled from synthetic oligonucleotides and/or polymerase chain reaction products, and the fragment was inserted into the transfer vector pCyA-20 to generate the pCyA-20-TMEP-B plasmid (pc-DNA-TMEP-B). The production of unmodified (TMEP-B) and modified (TMEP-Bmod) mRNAs was carried out from the plasmid containing the TMEP multiepitope (pcDNA-TMEP-B) under the control of T7 polymerase (Trilink BioTechnologies, Inc.; San Diego, CA, USA) [6].

Detection of Unmodified/Modified TMEP-B mRNA Expression by Flow Cytometry
TMEP-B and TMEP-Bmod expression in DCs was determined by flow cytometry 24 h after electroporation, using a specific antibody against the FLAG tag sequence located at the C-terminus of the TMEP-B sequence [6,7,26]. Cells were collected and washed with PBSx1 supplemented with 0.5% BSA and 0.1% sodium azide, before being centrifuged at 2000 rpm for 5 min. After surface staining (see above), cells were fixed/permeabilized with BD Cytofix/Cytoperm (BD Biosciences) at 4 • C for 20 min. Cells were then washed with PermWash buffer 1× (PW) (BD Biosciences) and centrifuged at 2000 rpm for 5 min. A blocking step was performed using an FcR Blocking Reagent (Milteny Biotec, Bergisch Gladbach, Alemania) for 30 min at 4 • C. After blocking, cells were washed twice with PW 1× and centrifuged at 2000 rpm for 5 min. Next, resuspended cells were incubated with 5 µg/mL of the monoclonal antibody anti-FLAG M2 (Sigma-Aldrich, Burlington, MA, USA) in PW 1× at 4 • C in the dark for 45 min. The cells were then washed twice with PW 1× and the secondary anti-mouse IgG (H + L) Alexa488 antibody diluted (1:400, Invitrogen, Carlsbad, CA, USA) in PW 1× was added to the cells at 4 • C for 45 min in the dark. After incubation, cells were washed twice with PW 1× and fixed with 1% formaldehyde. Samples were acquired with a FACSCanto II flow cytometer (BD) and data analyses was completed using FlowJo (Version 10.4.2; Tree Star, Ashland, OR, USA).

Blocking PD1 Expression on T-Cells
PD1 expression on T-cells was blocked by incubating with 20 µg/mL Nivolumab (anti-PD1, MedChemExpress, NJ, USA) for 30 min at 37 • C. After incubation, cells were washed twice with PBSx1 supplemented with 0.5% BSA and 0.1% sodium azide, then washed once with PBSx1 before being centrifuged at 1500 rpm for 5 min. Next, cells were incubated with Fc Block (Milteny Biotech, Bergisch Gladbach, Alemania) for 30 min at 4 • C and then washed with PBSx1 supplemented with 0.5% BSA and 0.1% sodium azide before being centrifuged at 1500 rpm for 5 min. The cells were then subjected to surface staining using anti-nivolumab PE (Anti-Human IgG4 pFc, Abcam, Cambridge, United Kingdom), anti-PD1 PE-Cy7 (Clone: EH12.1, BD Pharmingen, San Jose, CA, USA), and CD3 PERCP (Clone: HIT3a, BD Pharmingen) [27]. The corresponding isotypes and tube with cells without previous nivolumab incubation were used as controls. PD1 expression and nivolumab binding through human IgG4 were evaluated by flow cytometry using FACSCanto II (BD Biosciences) and analyzed using FlowJo (Tree Star, Ashland, OR, USA).

Co-Cultures: Electroporated DCs with Autologous Lymphocytes
The stimulatory function of the electroporated DCs was assessed by their ability to induce in vitro proliferation of autologous nonadherent PBMCs used as a source of enriched T-cells. These T-cells (2 × 10 5 cells/well) were incubated in X-VIVO 10 medium with the DCs (6 × 10 4 cells/well) previously electroporated with TMEP-B or TMEP-Bmod mRNA, and then they were seeded in 96 round-well plates at 37 • C in a humid 5% CO 2 atmosphere at a ratio of 1 DC to 3 T-cells. Mock electroporated DCs were used as negative controls. To determine the roles of Nivolumab (Anti-PD1) and Vesatolimod (TLR7 agonist) on T-cell proliferation, we added 20 µg/mL of Nivolumab (MedChemExpress) and 1000 nM of Vesatolimod into the DC/T-cells co-culture either separately or together. Both agents were added when setting up the co-cultures that also contained the cytokine cocktails.
To assess T-cell proliferation, these were stained with carboxyfluorescein succinimidyl ester (CFSE) using the Cell Trace CFSE proliferation kit (Invitrogen) according to the manufacturer's instructions. T-cell proliferation was measured by flow cytometry of CFSE dilution after 6 days of co-culture and expressed as the percentage of CFSE low cells after 6 days of co-culture under the different conditions.

Statistical Analysis
Cytometry results are shown as the means ± standard deviation (SD) of percentages or as the mean fluorescence intensity (MFI). Data analysis and comparison of each parameter relied on Student t-tests and nonparametric Wilcoxon signed rank tests, as appropriate. Statistical analysis was performed using GraphPad Prism, version 5 (La Jolla, CA, USA). For all analyses, the level of significance was set at * p < 0.05, ** p < 0.01 and *** p < 0.001.

Cytokines and Chemokines That Promote Viral Control Increase after TMEP-B or TMEP-Bmod Are Combined with Nivolumab or Vesatolimod
The secretions of IFNγ and MIP-1β increased, as did IP-10 to a lesser degree, after combining TMEP-B or TMEP-Bmod with vesatolimod ( Figure 5C). This effect on IFNγ secretion was also observed for DCs transfected with TMEP-B or TMEP-Bmod when combined to nivolumab alone or with both nivolumab and vesatolimod ( Figure 5C). The use of nivolumab in combination with different agents had a significant impact on IFNγ secretion ( Figure 5C). This effect of nivolumab was also seen for MIP-β secretion ( Figure 5C). In the case of IP10 secretion, this effect was only seen when combining nivolumab with TMEP-B (p = 0.0078) and compared with TMEP-B alone or TMEP-B plus vesatolimod (p = 0.0156) ( Figure 5C).

Discussion
The development of an effective therapeutic vaccine against HIV depends not only on understanding the complexity of the immune responses against the virus but also its adaptation to selective pressure exerted by the host. In this study, we evaluated an immunogen against HIV-1 that encodes a multiepitopic T-cell protein, which has been named TMEP-B. In previous studies, vectoring TMEP-B in both DNA and MVA enhanced HIV-1 specific T-cell responses, supporting its potential utility in prime/boost strategies for HIV therapy [6]. In the present study, we have evaluated the effect of this TMEP-B immunogen on HIV-specific T-cells immune responses, using unmodified and modified mRNA vectors.
TMEP-B and TMEP-Bmod similarly increased CD86, CD80, CD83, and FLAG expression levels, suggesting that the chemical modification made in the mRNA did not affect DC maturation or mRNA translation. Similar results have been obtained in a recently published study using HEK293T cell line [26]. In this, FLAG expression determined by flow cytometry or Western blot at different time-points was higher when transfected with TMEP-B, suggesting that differences in FLAG expression depend on the technique, experimental approach, and cell type used, as well as the time in which protein expression is determined. This further corroborates our finding that chemical modification of mRNA did not seem to improve translation. In addition, CCR7 expression was increased in DCs electroporated with any of our mRNAs, showing the potential impact of each immunogen on DC migration to lymph nodes.
Analysis of how mRNAs affected T-cell proliferation revealed that both TMEP-B and TMEP-Bmod can increase proliferation. This was also related to an extremely mild in vitro increase in IL2 secretion. We found no differences in T-cell proliferation after transfecting DCs with TMEP-B or TMEP-Bmod, which could be explained by the method used to transfect cells. When electroporation is used for transfection, most of the material goes straight into the cytosol, thereby avoiding degradation in the endocytic pathway [13].
It should also be noted that mRNA vaccines delivered by lipid nanoparticles would be more relevant than electroporation for both in vitro and in vivo human experimentation. Therefore, we are now formulating our mRNA TMEP-B in lipid nanoparticles to increase their efficiency, expression, and stability for further testing.
TMEP-B and TMEP-Bmod both stimulated cytokine and chemokine secretion. Indeed, the IFNγ secretion important for viral control was similar using either TMEP-B or TMEP-Bmod, suggesting that this was probably independent of nucleoside modification in the mRNA. A recent publication of the impact of TMEP-B and TMEP-Bmod on cytokine and chemokine secretion notably found an increase in IFIT1 and IFIT2 secretion (Interferoninduced protein) at 6 h post-transfection when using RT-qPCR. Such data would indirectly corroborate the observed increase in IFNγ levels. However, they found differences when using TMEP-B and TMEP-Bmod for IFIT1 and IFIT2 secretion, with the TMEP-B triggering higher levels of IFIT1 than TMEP-Bmod in the THP-1 cell line and enhancing the expression of IFIT2 [26]. In our head-to-head comparison of both mRNAs, we found higher levels of certain cytokines and chemokines when using TMEP-B (e.g., IP10, MIP-1α, and MIP-1β). These have been described as major HIV suppressive factors produced by CD8+ T-cells [28]. Our data therefore suggest that other elements of mRNA design, not the chemical modification, could be involved in the observed immunogenic effects. Other studies performed in non-human primates have also showed that the optimization of non-coding mRNA elements could improve the immunogenicity and protective efficacy of mRNA vaccines [29,30]. This could be the case for TMEP-B mRNA in this research.
Adjuvants or immunomodulators given systemically could be used in combination with TMEP-B to improve antigen presentation and the specific host immune response. We used vesatolimod, a promising candidate that could combine with other immunotherapies to achieve a functional cure of HIV. Our results show that vesatolimod use in combination with the mRNAs led to increased T-cell proliferation for both CD4+ and CD8+ T-cells. Vesatolimod also favored IFNγ secretion and the production of other cytokines and chemokines (e.g., IP-10 or MIP-1β). It could be speculated that various cellular antiviral responses are induced upon the activation of TLR7, and that this includes the release of inflammatory cytokines. Furthermore, it is possible that TLR7 activation by vesatolimod could promote cross-presentation via antigen-presenting cells to enhance the CD8+ T-cell responses. Vesatolimod is described as a modulator of HIV-1 that activates immune cells in vitro. It has been described that combining a TLR-7 agonist with HIV peptides (derived from Gag, Pol, Env, and Nef) can increase CD8+ T-cell degranulation, cytokine production, and cytolytic activity [21]. Studies in non-human primates have demonstrated the ability of TLR7 agonists to induce potent immune responses with increased IFN-regulated antiviral gene expression [31]. Elsewhere, it has been shown that the combination of Ad26/MVA vaccination and TLR7 stimulation can improve virologic control and delayed viral rebound after interruptions to antiretroviral treatment [20]. Recent clinical trials of Vesatolimod in HIV infected individuals have shown that this produced a consistent cytokine response, interferon-stimulated gene expression, and lymphocyte activation [32,33].
The other immunomodulatory agent evaluated in this study was nivolumab, an inhibitor of the PD1/PDL1 pathway. Different studies have associated PD1 expression on the T-cells of HIV infected patients with eventual T-cell exhaustion and disease progression [34,35]. CD4+ T-cells expressing immune checkpoint inhibitors have also been found to be enriched in latent HIV-1, suggesting that PD1 is important in establishing HIV latency [22,25]. Thus, we envisage that using nivolumab during antigen presentation at the immune synapse could be used to rescue T-cells from exhaustion and anergy, while also avoiding the undesirable systemic effects of blockade and possibly boosting antivirus immunity. In fact, the expression, secretion, and binding of these soluble molecules (PD1/PDL1) after mRNA electroporation, favoring T-cell multifunctionality, has been previously demonstrated [23]. Our results showed that combining nivolumab with TMEP-B increased not only the proliferation of HIV-specific T-cells (both CD4+ and CD8+) and cytokines (IFNγ and IP10) but also that of other cytokines and chemokines with antiviral effects (e.g., MIP-1β). These data suggest that nivolumab improves the specific immune response against HIV infection. Studies in non-human primates with the simian immunod-eficiency virus have shown that PD1 blockade with an antibody led to a rapid expansion of virus-specific CD8+ T-cells with improved functional quality, improving B-cell proliferation and in increasing envelope-specific antibodies to the virus, thereby enhancing both cellular and humoral immune responses [36]. Other studies in non-human primates have shown improved antiviral function of CD8+ T-cells and B cells, together with a reduction in the size of the viral reservoir that led to improved control of viral rebound after ART interruption [36][37][38].
Finally, combining both immunomodulatory agents, vesatolimod and nivolumab, in our experiments did not increase the levels of CD4+ and CD8+ T-cell proliferation compared with the use of either agent alone. Research must now investigate the effects of both agents to determine the impact of our strategy on T-cell polyfunctionality.
Overall, we have shown that combining mRNA-based vaccines with immunomodulatory agents can enhance immunogenicity and increase the specific cellular immune response against HIV infection. Our findings suggest that the proposed combination approach represents a promising strategy that could deliver functional cure of HIV. Moreover, in the future our experimental approach could be used as a helpful platform to screen more and different immunogenic combinations. Further research is clearly warranted to tackle these possibilities.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/vaccines11020286/s1, Figure S1: Efficiency of the monocytes yield and MDDC generation from the PBMCs of HIV infected patients; Figure S2: Antibodies used for flow cytometry; Figure S3: Phenotype of MDDCs obtained from HIV infected patients; Figure S4: Analysis of CCR7 expression on DCs from HIV infected patients; Figure S5: Study of cytokines secretion in the co-cultures of DC-T cell from HIV infected patients; Figure S6: Study of cytokines secretion in absence or presence of Vesatolimod.  Institutional Review Board Statement: Human blood was collected from chronic HIV infected individuals. This study was reviewed and approved by the institutional Ethics review board (HCB/2018/0305).

Informed Consent Statement: Written informed consent was obtained from all study participants.
Data Availability Statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.