Plasmids encoding the light and heavy chain of the mouse αDEC205 antibody were kindly provided by Dr. Michel C. Nussenzweig (The Rockefeller University, New York, USA). The plasmid encoding the heavy chain of the mouse DEC205 fused to eight HIV-1 epitopes was previously described and contains the following epitopes: p6 (32-46), p17 (73-89), pol (785-799), gp160 (188-201), rev (11-27), vpr (65-82), vif (144-158), and nef (180-194) (39).

The production of αDECHIVBr8 mAb [original clone NLDC145 (24)] was performed after transient transfection of human embryonic kidney (HEK) 293T cells (ATCC, CRL-11268) exactly as described elsewhere (33). Briefly, 293T cells were cultured in 150 mm plates (Sarstedt) under standard conditions in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 1% (v/v) antibiotic-antimycotic (Invitrogen), 1% (v/v) l-glutamine (Invitrogen), and 5% (v/v) Ultra low IgG Fetal Bovine Serum (Invitrogen). When cell confluence reached 70%, 293T cells were transfected using 10 μg of the plasmids encoding the light and the heavy chains in the presence of 150 mM NaCl and 0.45 mg/mL polyethyleneimine (PEI) (Sigma Aldrich). After 7 days in culture at 37°C with 5% CO₂, the culture supernatants containing secreted antibodies were collected by centrifugation at 1,000 x g for 30 min at 4°C and filtered through 0.22 μM membrane. The chimeric αDECHIVBr8 mAb was precipitated by addition of ammonium sulfate (Sigma Aldrich) to 60% of the total culture volume, and resuspended/dialyzed overnight against PBS at 4°C. After purification by affinity chromatography with protein G beads column (GE Healthcare), fusion mAb was dialyzed against PBS, resolved on a SDS-12% polyacrylamide gel, quantified, and stored at −20°C until use.

Female BALB/c (H-2^d) mice with 6-to 8-week old were purchased from Centro de Desenvolvimento de Modelos Experimentais para Medicina e Biologia (CEDEME)- Brazil. Mice were housed and manipulated under specific-pathogen-free (SPF) conditions at the animal care facility of the Division of Immunology, Federal University of São Paulo (UNIFESP).

Groups of six mice were immunized twice, 2 weeks apart, with 4 μg of αDECHIVBr8 mAb by intraperitoneal (I.P) route in the presence of the following adjuvants: 50 μg of poly(I:C) (Invivogen), 20 μg of Monophosphoryl Lipid A (MPL) (Invivogen), or 10 μg of CpG ODN 1826 (Invivogen). The amount of adjuvants used was previously determined (53). Control groups were immunized with 4 μg of αDECHIVBr8 in the absence of adjuvant or with PBS only.

Fifteen and sixty days after the administration of the second dose (boost), mice were deeply anesthetized by ketamine/xylazine solution (300 and 30 mg/kg, respectively) and mesenteric lymph nodes and the spleen were aseptically removed. After obtaining single cell suspensions, cells were washed in 10 mL of RPMI 1640 (Gibco). Splenic red blood cells were lysed with 1 mL of ACK solution (150 mM NH₃Cl, 10 mM KHCO₃, 0.1 mM EDTA) for 2 min at room temperature. After two additional washes with RPMI 1640, splenocytes and lymph node cells were then resuspended in R10 (RPMI supplemented with 10% of fetal bovine serum, 2 mM L-glutamine, 1% v/v vitamin solution, 1mM sodium pyruvate, 1% v/v non-essential amino acids solution, 40 μg/mL of Gentamicin, 5 x 10⁻⁵ M 2-mercaptoetanol (all from Gibco) and 20 μg/mL of Cyprofloxacin (Ciprobacter, Isofarma). The viability of cells was evaluated using 0.2% Trypan Blue exclusion dye to discriminate between live and dead cells. Cell concentration was estimated with the aid of a cell counter (Countess, Invitrogen) and adjusted in cell culture medium.

One million splenocytes were incubated for 48 h in the presence of pooled HIV-1 peptides (5 μM) or medium alone as negative control. Culture supernatants were harvested and stored at −20°C until analysis. IL-2, IL-4, IL-6, IL-10, IL-17, IFNγ, and TNFα were detected simultaneously using mouse Th1/Th2/Th17 cytokine bead array (CBA) kit (BD Pharmingen), according to the manufacturer's instructions. The range of detection was 20–5,000 pg/mL for each cytokine.

The ELISpot assay was performed using mouse IFNγ ELISpot Ready-SET-Go! (eBiosciences) according to manufacturer's instructions. Splenocytes from immunized mice were obtained as described and assayed for their ability to secrete IFNγ after in vitro stimulation with individual or pooled HIV-1 peptides (5 μM) or medium alone as negative control. Spots were counted using an AID ELISPOT Reader System (Autoimmun Diagnostika GmbH, Germany). The number of IFN-γ producing cells/10⁶ splenocytes was calculated after subtracting the negative control values and the cutoff was 15 SFU per million splenocytes.

To analyze HIV-specific T cell expansion, proliferation, and cytokine production, splenocytes from immunized mice were labeled with carboxyfluorescein succinimidyl ester (CFSE) (54). In summary, freshly isolated splenocytes were resuspended (50 × 10⁶/mL) in PBS and labeled with 1.25 μM of CFSE (Molecular Probes) at 37°C for 10 min. The reaction was quenched with RPMI 1640 supplemented with 10% FBS (R10) and cells were washed/resuspended with R10. Cells were cultured in 96-well round-bottomed plates (5 × 10⁵/well in triplicate) for 5 days at 37°C and 5% CO₂ with medium alone or with pooled HIV-1 peptides (5 μM). After 4 days, cells were restimulated with pooled HIV-1 peptides (5 μM) in the presence of 2 μg/mL anti-CD28 (BD Pharmingen) and Brefeldin A- GolgiPlugTM (BD Pharmingen) for further 12 h. After the incubation period, cells were washed with FACS buffer (PBS with 0.5% BSA and 2 mM EDTA) and surface stained with anti-mouse CD3 APCCy7 (clone 145-2C11), CD4 PerCP (clone RM4-5), and CD8 Pacific Blue (clone 53-6.7) monoclonal antibodies for 30 min at 4°C. Cells were fixed and permeabilized using Cytofix/Cytoperm™ kit (BD Pharmingen), according to manufacturer's instructions. After permeabilization, cells were washed with Perm/Wash buffer (BD Biosciences) and stained with anti-mouse IL2 PE (clone JES6-5H4), TNFα PECy7 (clone MP6-XT22), and IFNγ APC (clone XMG1.2) monoclonal antibodies for 30 min at 4°C. Following staining, cells were washed twice and resuspended in FACS buffer. All antibodies were from BD Pharmingen. Samples were acquired on a FACSCanto II flow cytometer (BD Biosciences) and then analyzed using FlowJo software (version 9.9, Tree Star, San Carlo, CA). To analyze cellular polyfunctionality, we used the Boolean gate platform (FlowJo software) to create combinations of the three cytokines (IL-2, TNFα, and IFNγ) within the CFSE^low population (cells that have undergone at least one cycle of division) resulting in seven distinct patterns. Polyfunctionality was defined as the ability of cells to exert at least two functions. The gating strategy, illustrated using data from one representative experiment, is shown in Figure S1. The frequencies of cytokine producing cells were calculated by subtracting the frequency of cells that were stimulated in vitro with HIV peptides by the frequency of the cells that were cultured in the presence of medium alone (background). For each experiment performed, unstained and all single-color controls were processed to allow proper compensation.

Mice were immunized once with 4 μg of αDECHIVBr8 mAb combined with the different adjuvants (poly(I:C), MPL or CpG ODN 1826). After 12 h, splenocytes were stained with biotinylated anti-mouse CD3 (clone 145-2C11), CD19 (clone 1D3), and CD49b (clone DX5). After 30 min, cells were washed with FACS buffer and stained with streptavidin APCCy7, anti-mouse CD11c APC (clone HL3), IAIE PE (clone 2G9), CD8 Pacific Blue (clone 53-6.7), CD40 FITC (clone 3.23), CD80 PerCP (clone 16-10A1), and CD86 PECy7 (clone GL1). Samples were acquired on a FACSCanto II flow cytometer (BD Biosciences) and then analyzed using FlowJo software (version 9.9, Tree Star, San Carlo, CA). For each experiment performed, unstained and all single-color controls were processed to allow proper compensation. Three million events were acquired in a live lymphocyte gate.

Statistical significance (p-value) was calculated by Two-way ANOVA followed by Bonferroni post hoc test or unpaired t-test (different time points comparison). Statistical analysis and graphical representation of data was performed using GraphPad Prism version 7.0 software.

To examine the effect of different adjuvants on HIV-specific cellular immune response, mice were immunized with two doses of αDECHIVBr8 mAb in the presence of the TLR agonists poly(I:C), MPL or CpG ODN 1826. Fifteen or Sixty days after the boost, splenocytes from immunized mice were incubated with pooled HIV-1 peptides to analyze specific cytokine production. First, we evaluated IFNγ production by ELISpot assay (Figure 1A). We observed that 15 days after the boost splenocytes from mice immunized with αDECHIVBr8 mAb combined with poly(I:C) presented higher number of specific IFNγ producing cells (716 SFU/10⁶ cells) when compared to the groups immunized in the presence of MPL or CpG ODN 1826 (404 and 286 SFU/10⁶ cells, respectively). Moreover, a significant difference was observed between MPL and CpG ODN 1826 groups (Figure 1A, left). Sixty days after the boost, we detected the same profile albeit with lower magnitude. Mice immunized with αDECHIVBr8 combined with poly(I:C) displayed 514 SFU/10⁶ cells while MPL and CpG ODN 1826 presented 284 and 142 SFU/10⁶ cells, respectively (Figure 1A, right). A comparison between 15 and 60 days revealed a significant decrease in the magnitude for poly(I:C) (p < 0.001), CpG ODN 1826 (p < 0.001), and MPL (p < 0.01) immunized groups. Splenocytes from mice immunized with αDECHIVBr8 in the absence of adjuvant or PBS (control groups) presented negligible numbers of IFNγ producing cells.

We also analyzed the cytokine profile by CBA assay using supernatant culture of splenocytes stimulated with pooled HIV peptides. Splenocytes from mice that received αDECHIVBr8 combined with poly(I:C) produced higher levels of IFNγ when compared to MPL or CpG ODN 1826, corroborating the ELISpot findings (Figure 1B). Interestingly, in the poly(I:C) adjuvanted group IFNγ production was even higher 60 days after the boost when compared to the 15 days time point (p < 0.001). Poly(I:C) also induced superior IL-2 production 15 days after the boost (Figure 1C, left). However, 60 days after the boost, IL-2 production significantly decreased in the group immunized with αDECHIVBr8 plus poly(I:C) and increased in the group that received the mAb in the presence of MPL (p < 0.001) (Figure 1C, right). IL-2 production by the group that received the mAb with CpG ODN 1826 slightly increased 60 days after the boost when compared to 15 days time point (p < 0.001). Regarding TNFα production 15 days after the boost, we observed that αDECHIVBr8 mixed with MPL produced the highest levels (Figure 1D). TNFα levels increased 60 days after the boost for the poly(I:C) group (p < 0.001) and decreased for the CpG ODN 1826 group (p < 0.05). No difference was observed for the MPL immunized group. Inflammatory IL-6 (Figure 1E) was higher in the group immunized with αDECHIVBr8 plus MPL 15 days after boost, but at the later time point the levels of this cytokine significantly decreased (p < 0.01). In contrast, 60 days after the boost with mAb and poly(I:C), IL-6 (p < 0.01) production increased considerably. IL-10 (Figure 1F) was superior in the poly(I:C) immunized group in both time points followed by MPL immunized group. However, after 60 days, IL-10 production decreased in the MPL (p < 0.001) and in the CpG ODN 1826 (p < 0.05) groups. Of note, IL-4 and IL-17 production was below the assay detection limit (data not shown). Taken together, these results indicate that different adjuvants induce a type 1 immune response when multiple HIV-antigens are delivered to CD8α⁺ DCs by the endocytic receptor DEC205.

In an attempt to evaluate HIV-specific CD4⁺ and CD8⁺ T cell responses, splenocytes from immunized mice were labeled with CFSE and pulsed in vitro with HIV-1 peptides. After culture, the frequency of CD3⁺CD4⁺CFSE^low (Figure 2A) and CD3⁺CD8⁺CFSE^low (Figure 2B) were evaluated by flow cytometry. Fifteen days after boost, splenocytes from mice that received αDECHIVBr8 along with poly(I:C) presented higher frequency of proliferating CD4⁺ (9.96%) and CD8⁺ (5.90%) T cells when compared to MPL immunized groups (6.83 and 4.86%, respectively). In contrast, CpG ODN 1826 displayed the lowest frequency of proliferating T cells. The same profile was observed 60 days after the boost, with the group that received αDECHIVBr8 plus poly(I:C) displaying higher CD4⁺ (11.30%) and CD8⁺ (6.17%) specific proliferation when compared to MPL (CD4⁺CFSE^low 4.86% and CD8⁺CFSE^low 2.47%) or CpG ODN 1826 (CD4⁺CFSE^low 3.60% and CD8⁺CFSE^low 1.31%) (Figures 2A,B right, respectively). Comparative analyses showed significant difference on the frequency of CD4⁺CFSE^low cells between 15 and 60 days only for the group that received αDECHIVBr8 plus MPL (p < 0.05). Regarding the CD8⁺ T cell compartment (CD8⁺CFSE^low cells), a significant difference was observed for MPL (p < 0.05) or CpG ODN 1826 (p < 0.01) groups. In contrast, mice immunized with αDECHIVBr8 in the presence of poly(I:C) displayed similar frequency of proliferating CD4⁺ and CD8⁺ T cells in all time points. To further characterize the functional profile of antigen-specific T cells, we assessed the ability of single cells to proliferate and produce the cytokines IFNγ, TNFα, and IL2 individually or simultaneously. The flow cytometry profile demonstrated that immunization with αDECHIVBr8 mAb along with poly(I:C) induced higher frequency of CD4⁺ T cells that proliferated and produced IFNγ⁺IL2⁺TNFα⁺ or IFNγ⁺TNFα⁺ simultaneously or only one cytokine (IFNγ or TNFα) 15 or 60 days after the boost (Figures 3A,B, respectively). Interestingly, for the poly(I:C) and MPL groups 60 days after the boost, the frequency of polyfunctional CD4⁺ T cells that proliferated and produced IFNγ, TNFα, and IL-2 simultaneously decreased, leading to an increase in the double or single cytokine producers (Figure 3–pie charts). Moreover, αDECHIVBr8 mixed with poly(I:C) also displayed higher frequency of proliferating CD8⁺ T cells that produce IFNγ or TNFα 15 or 60 days after the boost when compared with other groups (Figures 3C,D, respectively). Similarly to what was observed with the CD4 compartment at the later time point (60 days), there was also a shift in the CD8⁺ T cell polyfunctional profile in all groups when compared to 15 days after the boost; the frequency of three cytokine producing cells diminished while the single cytokine producers augmented (Figure 3 pie charts). Altogether, these results demonstrated that immunization with two doses of αDECHIVBr8 along with poly(I:C) induced higher and long-lasting specific polyfunctional CD4⁺ and CD8⁺ T cells responses.

To assess the breadth of T cell responses, splenocytes from immunized mice were incubated with single HIV-1 peptides present in the fusion vaccine and the number of IFNγ producing cells was determined by ELISpot. Fifteen days after last dose (Figure 4A), all adjuvants tested were able to induce positive responses against all peptides, albeit at different magnitudes (poly(I:C) > MPL > CpG ODN). At a later time point (Figure 4B), poly(I:C), and CpG ODN adjuvanted groups sustained IFNγ production against all peptides (head-to-head comparison in Figures S2A,C). On the contrary, in the MPL group, the magnitude of the response was more significantly reduced when we compared the 15 and 60 days time points (Figure S2B). Thus, multiepitope in vivo targeting to DEC205⁺ DCs when combined with poly(I:C) induced broad, potent and long-lasting T cell responses.

To further characterize phenotypic differences among the adjuvants, we compared the maturation status of splenic DCs after in vivo administration of the mAb combined with poly(I:C), MPL or CpG ODN 1826. The gating strategy, illustrated using data from one representative experiment, is shown in Figure S3. Twelve hours after injection, CD11c⁺CD8α⁺ DCs from poly(I:C) group considerably up-regulated the expression of CD80 compared to other groups (Figures 5A,B). CpG ODN 1826 slightly increased CD80 expression only when compared to MPL. However, none of the adjuvants up regulated CD80 expression on CD11c⁺CD8α⁻ DCs. Furthermore, poly(I:C) was the only adjuvant to significantly up regulate CD86 expression in both DCs subsets (Figures 5C,D). Similarly, we observed a significant increase in the MFI of CD40 molecule by poly(I:C) in both DCs subsets when compared to other adjuvants (Figures 5E,F). In addition, to assess whether DC activation could occur earlier than 12 h, we analyzed the expression of costimulatory molecules 6 h after injection, and observed the same pattern of CD80, CD86, and CD40 expression in both DCs subsets (Figures S4A–C, respectively). We also analyzed the activation profile on mesenteric lymph nodes and the same pattern of expression was observed (data not shown). Taken together, these results strength the idea that poly(I:C) is a superior adjuvant than MPL or CpG ODN 1826 since it up regulates costimulatory molecules in both splenic DCs subsets (CD8α⁺ and CD8α⁻).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.