The sequences of HIV-1 epitopes selected for this study were previously described by Fonseca et al. (16) and are the following: 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) (Table 1). These epitopes were derived from the previously described DNA vaccine HIVBr18 (18, 19) and comprise the eight mentioned epitopes (HIVBr8) that can bind to I-Ad and are recognized by T cells from immunized BALB/c mice. The epitopes were assembled in tandem and are separated by GPGPG at C and N termini to avoid the creation of junctional epitopes that may interfere with processing and presentation (43).

The HIVBr8 nucleotide sequence was codon optimized, and a Kozak sequence was included at the 5′ end to improve mammalian expression. The artificial gene (Genscript) was cloned between the HindIII and XhoI restriction sites of the pVAX1 vector (Invitrogen) to generate the pVAXHIVBr8 plasmid that was amplified using DH5α cells. The pVAXHIVBr8 was purified using the Endofree Plasmid Giga Kit (Qiagen) according to the manufacturer’s instructions. The yield and quality of purified DNA was determined by spectrophotometry at 260 nm and confirmed by agarose gel electrophoresis.

Plasmids encoding the light and heavy chains of the mouse αDEC205 antibody were kindly provided by Dr. Michel C. Nussenzweig (The Rockefeller University). The artificial HIVBr8 gene was produced (Genscript), digested from pUC57 vector, and cloned in frame with the carboxyl terminus of the heavy chain of the mouse DEC205 (clone NLDC145) between the 5′ XhoI and 3′ NotI sites. Large-scale preparation of plasmids pDECHIVBr8, empty pDEC (a negative non-fused control), and pDEC kappa (encoding the αDEC205 kappa light chain) were prepared using Maxi Plasmid Purification Kit (Qiagen), according to the manufacturer’s instructions. The yield and quality of purified DNA were determined by spectrophotometry at 260 nm and confirmed by agarose gel electrophoresis.

The chimeric αDEC and αDECHIVBr8 mAbs were produced and purified after transfection of human embryonic kidney 293T cells (ATCC, CRL-11268) exactly as described (35).

Approximately 1 µg of the αDEC and αDECHIVBr8 mAbs were run on 12% SDS-PAGE gels under reducing conditions, and subsequently transferred to nitrocellulose membranes (GE Healthcare) at 100 V for 90 min in transfer buffer (glycine 39 mM, Tris 48 mM, SDS 10% and methanol 20%, pH 8.3). After transfer, nitrocellulose membranes were blocked in PBS 0.02% Tween-20 (PBST), 5% non-fat milk, and 2.5% BSA, overnight at 4°C. Membranes containing the reduced mAbs were then incubated with peroxidase-labeled goat anti-mouse IgG Fc specific (1:5,000, Jackson Laboratories) plus peroxidase-labeled goat anti-mouse IgG kappa (1:3,000, SouthernBiotech) for 60 min at room temperature. After three washes with PBST, the reaction was developed using chemiluminescence (ECL kit, GE Healthcare) and captured on Kodak film.

Binding assays were performed using CHO cells expressing either the mouse DEC205 (CHOmDEC) or DCIR2 (CHOmDCIR2) receptors, kindly provided by Dr. Michel Nussenzweig (The Rockefeller University). Purified mAbs were diluted to 4, 2, or 1 µg/mL and incubated with the CHOmDEC or CHOmDCIR2 cells at 4°C for 30 min, exactly as described by Henriques et al. (35). Next, the cells were washed and incubated with anti-mouse IgG1-PE (clone A85-1, BD Biosciences) for 30 min at 4°C. Additionally, 4, 2, or 1 µg/mL of αDEC or αDECHIVBr8 mAbs were incubated with 5 million splenocytes at 4°C for 40 min and then incubated with anti-CD49b-biotin (clone DX5), anti-CD19-biotin (clone 1D3), anti-CD3-biotin (clone 145-2C11), Streptavidin-APCCy7, anti-CD11c-APC (HL3), anti-IAIE-FITC (clone 2G9), anti-CD8-Pacific Blue (clone 53-6.7), and anti-IgG1-PE (clone A85-1). All monoclonal antibodies were purchased from BD Biosciences. Fifty thousand events were acquired for the analysis of binding to CHO cells and 3 million for the analysis of binding to splenocytes. Samples were acquired using FACS Canto II flow cytometer (BD Biosciences) and analyzed using the FlowJo software (version 9.9, Tree Star, San Carlos, CA, USA).

The 6- to 8-week-old female BALB/c (H-2^d) mice were purchased from Centro de Desenvolvimento de Modelos Experimentais para Medicina e Biologia (CEDEME), Brazil. Groups of six animals were immunized with two doses—2 weeks apart—of 4 µg of αDECHIVBr8 mAb in the presence of 50 µg of adjuvant poly (I:C) (Invivogen) delivered intraperitoneally (IP) or subcutaneously (SC), or with two doses of 100 µg of the DNA vaccine pVAXHIVBr8 by intramuscular route (IM). The control groups were immunized with 4 µg of αDEC in the presence of 50 µg of poly (I:C) or with pVAX (empty vector). Furthermore, for heterologous prime-boost regimen, other groups received one dose of the mAb followed by one dose of DNA vaccine or vice versa. The control groups were immunized with one dose of αDEC mAb together with poly (I:C) followed by one dose with pVAX or vice versa.

Two weeks after the last immunization, mice were euthanized and spleens were removed aseptically. After obtaining single cell suspensions, cells were washed in 10 mL of RPMI 1640 (Gibco). Cells were then resuspended in R-10 [RPMI supplemented with 10% of fetal bovine serum (Gibco)], 2 mM L glutamine (Gibco), 10 mM Hepes (Gibco), 1 mM sodium pyruvate (Gibco), 1% v/v non-essential amino acids solution (Gibco), 40 µg/mL of gentamicin, 20 µg/mL of peflacin, and 5 × 10⁻⁵ M 2-mercaptoethanol (Gibco). 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.

Splenocytes from immunized mice were obtained as previously described and assayed for their ability to secrete IFNγ after in vitro stimulation with 5 µM of individual or pooled HIV-1 peptides using the ELISpot assay. The ELISpot assay was performed using mouse IFNγ ELISpot Ready-SET-Go! (eBiosciences) according to the manufacturer’s instructions. Spots were counted using an AID ELISpot Reader System (Autoimmun Diagnostika GmbH, Germany). 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) (19). 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 with R10 before resuspension in RPMI 1640. Cells were cultured in 96-well round-bottomed plates (5 × 10⁵/well in triplicates) for 5 days at 37°C and 5% CO₂ with medium only or pooled HIV-1 peptides (5 µM). After 4 days of incubation, cells were restimulated in the presence of 2 µg/mL anti-CD28 (BD Pharmingen), 5 µM of individual or pooled HIV-1 peptides and brefeldin A GolgiPlug™ (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-CD3 APCCy7 (clone 145-2C11), anti-CD4 PerCP (clone RM4-5), and anti-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 the manufacturer’s instructions. After permeabilization, cells were washed with Perm/Wash buffer (BD Biosciences) and stained intracellularly with anti-IL2 PE (clone JES6-5H4), anti-TNFα PECy7 (clone MP6-XT22), and anti-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 Carlos, CA, USA). To analyze cellular polyfunctionality, we used the Boolean gating platform (FlowJo software) to create several combinations of the three cytokines (IL-2, TNFα, and IFNγ) within the CFSE^low population resulting in seven distinct patterns. The percentages of cytokine-producing cells were calculated by subtracting background values. For each experiment performed, unstained and all single-color controls were processed to allow proper compensation.

Statistical significance (p-values) was calculated by using a two-way ANOVA and Bonferroni’s or one-way ANOVA and Tukey honest significant difference. Statistical analysis and graphical representation of data was performed using GraphPad Prism version 5.0 software.

Mice were housed and manipulated under SPF conditions in the animal care facilities of the Division of Immunology, Federal University of São Paulo (UNIFESP). This study was carried out in accordance with the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Brazilian National Law (11.794/2008). The protocol (number 3226180814) was approved by the Institutional Animal Care and Use Committee (CEUA) of Federal University of São Paulo.

In an attempt to induce a T cell response against HIV, we cloned eight CD4⁺ T cell epitopes in fusion with the heavy chain of the DEC205 receptor. αDECHIVBr8 and control αDEC205 mAbs were purified and analyzed in 12% SDS polyacrylamide gel under reducing conditions. Figure 1A shows an immunoblot in which both mAbs were transferred to a nitrocellulose membrane and incubated with anti-mouse total IgG and anti-mouse IgG kappa chain. Two bands that correspond to the heavy chain (~50 kDa) and to the light chain were detected. In the αDECHIVBr8 mAb preparation, we detected the light chain (~25 kDa) and also a band of ~70 kDa that corresponds to the heavy chain of αDEC205 fused with the HIVBr8 sequence. Next, we tested whether the αDECHIVBr8 mAb retained its binding capacity to either CHO cells expressing the mouse DEC205 receptor or to the CD11c⁺CD8α⁺ spleen DCs. Figure 1B shows that αDECHIVBr8 mAb was able to specifically bind to CHO cells expressing the mouse DEC205 receptor in a dose-dependent manner but not to CHO cells expressing the mouse DCIR2 receptor. The control αDEC205 mAb showed the same binding pattern. More interestingly, the αDECHIVBr8 mAb was able bind specifically to the murine CD11c⁺CD8α⁺ DCs but not to the CD11c⁺CD8α⁻ DCs, showing its specificity for DCs that express DEC205 in vivo. As expected, the control αDEC205 mAb also bound to the CD11c⁺CD8α⁺ DCs (Figure 1C). Taken together, these results showed that the αDECHIVBr8 mAb was successfully produced and retained its capacity to bind to murine DCs expressing DEC205.

We initially evaluated the cellular immune responses against the pooled HIV peptides in BALB/c mice immunized with one or two doses of αDECHIVBr8 mAb (4 µg) in the presence of poly (I:C) and compared to two doses of the pVAXHIVBr8 DNA vaccine (100 µg) (Figure 2A). Splenocytes from mice immunized with two doses of the αDECHIVBr8 mAb presented a higher number of specific IFNγ-producing cells when compared to mice immunized with two doses of the DNA vaccine (about 280 and 180 SFU/10⁶, respectively). In addition, no significant difference was observed when the groups that received one or two doses of the αDECHIVBr8 mAb were compared (Figure 2B). Notably, when specific cellular proliferation was evaluated, mice that received two doses of αDECHIVBr8 mAb displayed higher frequency of specific CD4⁺ (12%, Figure 2C) and CD8⁺ (8.5%, Figure 2D) T cells that proliferated when compared to all the other groups. Significantly, these were almost twofold higher than the numbers found in the group receiving two doses of pVAXHIVBr8 (6.6% CD4⁺ and 4.8% specific CD8⁺ T cells). Control groups that were immunized with αDEC mAb or pVAX plasmid did not show specific IFNγ production or T cell proliferation. Of note, the number of CD4⁺ and CD8⁺ T cells that proliferated in mice immunized with two doses of the αDECHIVBr8 mAb was higher than the number detected in mice immunized with just one dose. These results led us to conclude that two doses are more effective to induce higher immune responses.

Next, we decided to address if the route of immunization would alter the efficacy of the αDECHIVBr8 mAb immunization. For that purpose, mice were immunized with two doses of the αDEC or αDECHIVBr8 in the presence of poly (I:C) by intraperitoneal (IP) or subcutaneous (SC) route (Figure 3A). As shown in Figure 3B, IP immunization with the αDECHIVBr8 mAb was able to induce higher IFNγ-producing cells than the SC immunization. Similar results were obtained when we measured the percentage of specific proliferating CD4⁺ and CD8⁺ T cells (Figures 3C,D, respectively). Control mice immunized with αDEC205 mAb did not show significant production of IFNγ or proliferation independently of the route used. We subsequently characterized the profile of polyfunctional T cells. Using multiparameter flow cytometry, we detected antigen-specific T cells (CD4⁺ and CD8⁺) based on their ability to proliferate (CFSE dilution assay) and produce the effector cytokines IFNγ, TNFα, and IL2 simultaneously. Boolean combinations of proliferating and cytokine-positive populations indicated that immunization by IP route was most effective to induce higher percentage CD4⁺ T cells that proliferated and produced simultaneously IFNγ/IL2/TNFα or IFNγ/TNFα or TNFα only (Figure 3E). Also, IP immunization induced a higher percentage of CD8⁺ T cells that proliferated and produced IFNγ/IL2/TNFα simultaneously when compared to the group immunized by SC route (Figure 3F). We can conclude that immunization using two doses of the αDECHIVBr8 mAb by the IP route was better to induce T cell responses with greater magnitude and more polyfunctional than those induced by the pVAXHIVBr8 DNA vaccine encoding the same epitopes.

To test whether a heterologous prime-boost strategy would work with chimeric mAb, groups of mice were then immunized with one dose of αDECHIVBr8 mAb (prime) followed by one dose of the pVAXHIVBr8 DNA vaccine (boost), or vice versa, and compared to the homologous prime-boost immunization strategy (two doses of chimeric mAb or DNA vaccine) (Figure 4A). Two weeks after the boost, splenocytes from immunized mice were incubated with pooled peptides and specific IFNγ production was measured by ELISpot assay. We detected the highest numbers of IFNγ-producing cells against the pooled peptides in mice that received two doses of αDECHIVBr8 and in the group receiving pVAXHIVBr8 priming followed by αDECHIVBr8 mAb boosting (Figure 4B). Interestingly, mice primed with αDECHIVBr8 mAb and boosted with pVAXHIVBr8 showed a lower response when compared to the two previously described groups. Finally, mice immunized with two doses of pVAXHIVBr8 presented the lowest number of IFNγ-producing cells (416 SFU/10⁶ cells, Figure 4B). A similar pattern was observed when we analyzed the percent of CD4⁺ and CD8⁺ specific proliferation (Figures 4C,D, respectively): mice immunized with two doses of αDECHIVBr8 mAb displayed 8.23% of CD4⁺ and 7.49% of CD8⁺ T cell specific proliferation, while pVAXHIVBr8 (prime)/αDECHIVBr8 (boost) displayed 7.04 and 7.86%, respectively. Lower percentages were observed in mice immunized with two doses of pVAXHIVBr8 or αDECHIVBr8 mAb (prime)/pVAXHIVBr8 (boost). In addition, we characterized the phenotype and functionality of antigen-specific CD4⁺ and CD8⁺ T cells based on their ability to proliferate (CFSE^low) and produce IFNγ, TNFα, and IL2 individually or in combinations (Figure S1 in Supplementary Material). Figure 4E shows once more that immunization with two doses of αDECHIVBr8 and pVAXHIVBr8 (prime)/αDEC205-HIVBr8 (boost) were most effective to induce higher percentage of CD4⁺ T cells that proliferated and produced simultaneously IFNγ/TNFα or IFNγ only or TNFα only. When we analyzed the CD8⁺ T cells, two doses of pVAXHIVBr8 DNA vaccine showed a higher percentage of CD8⁺ T cell proliferating and producing only IFNγ when compared to the other groups (Figure 4F). By contrast, CD8⁺ T cells from mice immunized with two doses of αDECHIVBr8 and pVAXHIVBr8 (prime)/αDECHIVBr8 (boost) were able to proliferate and mainly produce TNFα. αDEC and pVAX immunized mice displayed negligible percentages of specific proliferating/cytokine-producing T cells. Extended comparative analysis revealed that αDECHIVBr8 and αDECHIVBr8 (prime)/pVAXHIVBr8 (boost) immunized mice displayed higher frequency of non-proliferating (CFSE^high) IFNγ⁺/IL2⁺/TNFα⁺ and CFSE^high IFNγ⁺/TNFα⁺-producing CD4⁺ T cells when compared to other groups (Figure S2A in Supplementary Material). Analysis of CD8⁺ T cell compartment demonstrated that αDECHIVBr8 (prime)/pVAXHIVBr8 (boost) was the most efficient strategy to induce CFSE^high IFNγ⁺/IL2⁺/TNFα⁺ and CFSE^high IFNγ⁺ cells, while αDECHIVBr8 immunization induced higher frequency of CFSE^high TNFα⁺ cells (Figure S2B in Supplementary Material). Taken together, these results showed that homologous immunization with αDECHIVBr8 mAb or heterologous pVAXHIVBr8 (prime)/αDECHIVBr8 (boost) were able to induce broad specific cellular responses, and polyfunctional CD4⁺ and CD8⁺ T cells that proliferated and produced effector Th1 cytokines to epitopes encoded by the chimeric mAb and the DNA vaccine.

In an attempt to improve the cellular immune response induced by the DNA vaccination alone and compare it to the homologous immunization with αDECHIVBr8 or with the heterologous pVAXHIVBr8 (prime)/αDECHIVBr8 (boost), we decided to administer one additional dose of either αDECHIVBr8 or pVAXHIVBr8. In this way, the homologous immunization groups received three doses of either αDECHIVBr8 or pVAXHIVBr8, while the heterologous immunization group received one dose of pVAXHIVBr8 followed by two boosters of αDECHIVBr8. Control groups received three doses of either αDEC or pVAX (Figure 5A). To assess the magnitude of the immune response, we evaluated specific IFNγ-producing cells in splenocytes from immunized mice stimulated with pooled peptides (Figure 5B). Once again, we observed no difference between animals immunized with three doses of αDECHIVBr8 and animals immunized with pVAXHIVBr8 once/αDECHIVBr8 twice. By contrast, the group immunized with pVAXHIVBr8 thrice presented a lower number of specific IFNγ-producing cells. In addition, as observed with the administration of two doses, CD4⁺ and CD8⁺ T cells (Figures 5C,D, respectively) from mice immunized with three doses of αDECHIVBr8, and one dose pVAXHIVBr8 followed by two doses of αDECHIVBr8 displayed higher T cell proliferation against the pooled HIV-1 peptides than three doses of pVAXHIVBr8. By contrast, mice immunized with three doses of pVAXHIVBr8 continued to present a lower percentage of proliferation when compared to the two previous groups. αDEC and pVAX immunized mice presented negligible numbers of IFNγ-producing cells and T cell proliferation. Boolean combinations of proliferating and cytokine-positive populations indicated that homologous immunization with αDECHIVBr8 (3×) or with pVAXHIVBr8 (1×)/αDECHIVBr8 (2×) induced polyfunctional CD4⁺ T cells that proliferated and produced simultaneously IFNγ/TNFα, or IFNγ, or TNFα only (Figure 5E). Similar results were observed when CD8⁺ T cells were analyzed (Figure 5F). CD4⁺ and CD8⁺ T cells from mice immunized with the controls αDEC and pVAX displayed negligible percentages of specific proliferating/cytokine-producing T cells. Taken together, these results indicate that an additional dose of pVAXHIVBr8 does not improve the magnitude of the T cell responses over immunization with αDECHIVBr8 mAb.

To evaluate whether immunization induces broad T cell responses, splenocytes from mice immunized with two doses of αDECHIVBr8 and pVAXHIVBr8 or heterologous prime-boost regimens (Figure 6A) were incubated with each of the eight individual HIV-1 peptides present in the chimeric mAb or DNA vaccine. We detected IFNγ-producing cells against all tested peptides. Immunization with two doses of αDECHIVBr8 mAb or with pVAXHIVBr8 (prime)/αDECHIVBr8 (boost) elicited significantly higher numbers of IFNγ-producing cells when compared to mice immunized with two doses of pVAXHIVBr8 or that received αDECHIVBr8 (prime)/pVAXHIVBr8 (boost) (Figure 6B).

In summary, ours results demonstrate that immunization of BALB/c mice with two or three doses of αDECHIVBr8 mAb or heterologous prime-boost with pVAXHIVBr8 followed by αDECHIVBr8 were able to induce specific immune responses with a higher number of IFNγ-producing cells, and polyfunctional CD4⁺ and CD8⁺ T cells that proliferated and produced effector Th1 cytokines.