The molecular immune modulator adenosine deaminase-1 enhances HIV specific humoral and cellular responses to a native-like HIV envelope trimer DNA vaccine

There is currently no prophylactic vaccine available for human immunodeficiency virus (HIV). Research efforts have resulted in improved immunogens that mimic the native envelope (Env) glycoprotein structure. Recently, a novel triple tandem trimer (TTT) platform has been used to generate a plasmid encoding Env immunogen (pBG505-TTT) that expresses only as trimers, making it more suitable for nucleic acid vaccines. We have previously demonstrated that adenosine deaminase-1 (ADA-1) is critical to the T follicular helper (TFH) function and improves vaccine immune responses in vivo. In this study, we demonstrate that co-delivery of plasmid-encoded adenosine deaminase 1 (pADA) with pBG505-TTT enhances the magnitude, durability, isotype switching and functionality of HIV-specific antibodies in a dose-sparing manner. Co-delivery of the molecular immune modulator ADA-1 also enhances HIV-specific T cell polyfunctionality, activation, and degranulation as well as memory B cell responses. These data demonstrate that pADA enhances HIV-specific cellular and humoral immunity, making ADA-1 a promising immune modulator for HIV-targeting vaccines.


Introduction
Human immunode ciency virus (HIV) remains a prominent global health threat for which no prophylactic vaccine is available.Although there have been many efforts to develop an HIV vaccine, its development has proven to be challenging.Due to the high sequence diversity and multiple routes of transmission of HIV, it is critical for an effective HIV vaccine to induce both circulating and mucosal broadly neutralizing antibodies (bNAbs) capable of neutralizing most HIV circulating strains.Properties of the HIV envelope (Env) protein, used for viral entry pose, additional challenges.HIV Env protein is extremely metastable and heavily glycosylated, resulting in very little access to immune recognition of neutralizing epitopes and subsequent immune evasion [1,2].
Vaccine attempts using monomeric gp120 subunits have failed to induce bNAbs and did not prevent infection [3][4][5], necessitating the development of improved envelope vaccine immunogens.Extensive research efforts have resulted in immunogens, including SOSIP trimers, that closely mimic the native Env glycoprotein structure and consistently induce autologous neutralizing responses [2,6,7].Previous studies using protein BG505-SOSIP vaccination have focused on humoral characterization with little cellular characterization, particularly CD8 + T cell responses [8,9].Recently, a novel triple tandem trimer (TTT) platform has been used to generate a plasmid encoding Env immunogen (pBG505-TTT) that expresses BG505 trimers, making it more suitable for nucleic acid vaccines [10].A recent study with another native-like Env immunogen MD39, showed that MD39-encoding DNA vaccination elicited enhanced Env-speci c humoral and cellular responses compared to MD39 protein.These results provided evidence that a DNA vaccine platform could be advantageous for driving anti-HIV responses over a protein subunit vaccine [11].
We have previously demonstrated that a novel molecular immune modulator, adenosine deaminase-1 (ADA-1) enhances the magnitude and durability of both cellular and humoral vaccine induced immune responses in vivo [12][13][14].This has been demonstrated across several vaccine antigens including SARS-CoV-2 [13,14] and monomeric gp160 HIV envelope [12] DNA vaccines.ADA-1 is necessary for normal immune function as exempli ed by the severe-combined immunode ciencies (SCID) that results from ADA-1 loss-of-function mutations [15].Pegylated ADA is used therapeutically as an enzyme replacement therapy for the treatment of ADA-SCID [16].The enzymatic function of ADA-1 is to catalyze the nonreversable deamination of adenosine into inosine therefore regulating both intracellular and extracellular adenosine concentrations.Independent of its enzymatic function, ADA-1 has also been shown to promote T cell proliferation and differentiation [17][18][19].Importantly, our lab has recently identi ed a novel function of ADA-1.We have demonstrated that ADA-1 prolongs TFH survival and induces TFH differentiation making it unique in boosting germinal center (GC) function resulting in increased quality of antigen speci c adaptive immune responses to vaccines [13,20].
TFH cells are necessary for germinal centers and are critical drivers of processes such as somatic hypermutation, a nity maturation and isotype switching in B cells.BNAbs against HIV exhibit high levels of somatic hypermutation and extensive a nity maturation [21].Circulating TFH frequencies and function have been correlated with bNAbs [21,22] and when administered passively, bNAbs against HIV have been shown to be protective against HIV acquisition in both humans and non-human primates (NHP) [23,24].In addition to antibody (Ab) neutralization, Fcγ receptor (FcγR)-mediated Ab function such as Ab dependent complement deposition (ADCD) has been shown to decrease HIV replication and may play a role in HIV protection [25][26][27][28][29][30].ADCD has been found to be primarily driven by non-neutralizing antibodies (nNAbs) in the context of HIV and such nNAbs have been found to act together with CD8 + T cells to confer heterosubtypic immunity in the context of other viral infections [31,32].CD8 + T cell function during chronic viral infections has been found to be sustained by TFH cells producing IL-21 [33].CD8 + T cells present in elite controllers of HIV infection have been found to establish immunological synapses with infected CD4 + T cells, leading to IFN-γ production, degranulation, and the elimination of the target cells [34].Vaccine strategies designed to enhance CD8 + T cell frequency and effector function have also demonstrated protective HIV immunity in NHP [35].These data demonstrate the importance of CD8 + T cell responses in an effective HIV vaccine and how vaccine strategies targeting TFH cells can drive both humoral responses and cellular responses that are critical for protective HIV immunity.
Since ADA-1 enhances TFH cell differentiation and function [20], and TFH cells are central to many aspects critical for an effective anti-HIV humoral and cellular response, we believe ADA-1 would serve as a promising immune modulator for HIV targeting vaccines.We therefore hypothesized that ADA-1 combined with the improved HIV envelope antigen pBG505-TTT would result in enhanced magnitude and quality of both humoral and cellular HIV speci c responses.
To evaluate this hypothesis, mice were immunized in the current study with pBG505-TTT alone or in combination with plasmid encoded ADA-1 (pADA).To understand how ADA-1 functions as an immune modulator in relation to more classically used adjuvants such as Alum and MF59, another cohort of mice were co-immunized with pBG505-TTT with or without pADA, recombinant BG505 protein (rBG505) and either Alum or an MF59-like adjuvant AddaVax.ADA-1 was found to enhance HIV speci c humoral responses as demonstrated by increased SOSIP-speci c antibody magnitude, durability, isotype switching and functionality.pADA co-immunization also resulted in enhanced HIV speci c cellular responses including increased T cell cytokine production, polyfunctionality, and activation as well as memory B cell responses.In comparison to other adjuvants, pADA was able to enhance Ab neutralization compared to alum while AddaVax induced robust Ab neutralization with no further enhancement with pADA.This study provides a proof of concept that ADA-1 can be used as an effective immune modulator for HIV nucleic acid-based vaccine strategies.
A codon-optimized DNA plasmid encoding murine ADA-1 (pADA) (NCBI gene 11486) was subcloned into the pVax expression vector and produced commercially (Genscript, Piscataway, NJ).In vitro studies revealed the expression of the ADA proteins after transfection of cell lines with the vaccine construct as previously described [12,38].
Protein production BG505 SOSIP.v8.1 proteins (untagged, his-tagged and avi-tagged) were expressed in HEK293F cells and puri ed by PG151 immunoa nity chromatography, similar to previously described [39].Brie y, HEK293F cells (Invitrogen, Cat:R79009) maintained in FreeStyle Expression Medium (Gibco) were transiently transfected with Env and furin expression plasmids in a 4:1 (w/w) Env to furin ratio.For transfection, the DNA mix was incubated with PEImax (Polysciences Europe GmBH, Eppelheim, Germany) in a 3:1 (w/w) PEImax to DNA ratio and then added to the supernatant of cells at a density of 0.8-1.2 million cells/mL.Five to seven days after transfection, supernatants were harvested, centrifuged and vacuum-ltered using 0.22 µm Steritops (Millipore, Amsterdam, The Netherlands).Proteins in the ltered supernatants were captured on PGT151-coupled CNBr-activated sepharose 4B beads (GE Healthcare) by owing at 0.5-1.0mL/min or overnight rolling incubation at 4°C.Subsequently, the beads were immobilized in Econo-Column chromatography columns (Biorad) and washed with three column volumes of a 0.5 M NaCl 20 mM Tris HCl pH 8.0 solution.After elution with 3 M MgCl2 pH 7.5, proteins were buffer exchanged to TN75 (75 mM NaCl, 20 mM Tris HCl pH 8.0) or PBS buffers using Vivaspin20 MWCO 100 kDa ultra ltration units (Sartorius, Gӧttingen, Germany).After puri cation, avi-tagged proteins were biotinylated using a BirA500 biotin-ligase reaction kit (Avidity, Aurora, USA) according to manufacturer's instructions.Excess biotin was removed by ultra ltration with Vivaspin6 MWCO 10 kDa lters (Sartorius, Gӧttingen, Germany).Protein concentrations were determined using a NanoDrop One spectrophotometer (ThermoFisher Scienti c) and molecular weight and extinction coe cient values calculated with the ProtParam webtool (Expasy).Puri ed proteins were lter sterilized and stored at -80°C until use.Untagged BG505 SOSIP.v8.1 protein was used for immunizations, his-tagged protein was used for ELISA assays, and avi-tagged biotinylated protein was used for memory B cell ow cytometry analysis.

Immunizations
Female BALB/c mice aged 6-8 weeks were immunized in the tibialis anterior (TA) muscle with 20-40 µl of the formulated vaccines.Mice were immunized with 1 or 5 µg pBG505-TTT alone or were coformulated with 10 µg pADA in the right TA muscle.Mice that were co-immunized with both pBG505-TTT and protein received 10 µg pBG505-TTT alone or with 10 µg pADA in the right TA muscle and 10 µg of recombinant BG505 protein (rBG505) co-formulated with protein adjuvants Alum (Aluminum hydroxide gel, Invivogen, Cat:vac-alu-250) or AddaVax (Invivogen, Cat:vac-adx-10) in the left TA muscle.Alum and MF59 were formulated at a 1:1 volume ratio with antigen (i.e.65.5 µL rBG505 + 65.5 µL Alum or AddaVax), per the manufacturer's recommendation.Mice that were immunized with protein alone received 10 µg rBG505 co-formulated with Alum or AddaVax in the right TA muscle (see Supplemental Fig. 1c).Control mice were immunized with 15 µg or 20 µg of empty plasmid vector (Pvax) to ensure an equal amount of DNA was administered across experimental groups.Immediately after DNA vaccine injection, in vivo electroporation was performed using the CELLECTRA device (Inovio Pharmaceuticals, Bluebell, PA).Animals were housed in a temperature-controlled, light-cycled, speci c-pathogen-free facility at Drexel University College of Medicine in accordance with protocols approved by Drexel University Institutional Animal Care and Use Committee.Previous studies immunizing male and female mice with pADA did not show any sex biases nor any species (C57BL/6 or BALBc) biases [13].

Mouse sacri ce, sample collection, and tissue harvest
At the time points shown in the in vivo study designs (Fig. 1a and Fig. 3a), mice were either bled via cheek bleed or sacri ced.At sacri ce, blood and spleens were collected.Blood collected via cheek bleed or cardiac puncture was collected into minicollect serum gel tubes (Grenier-Bio) and centrifuged at 16,000 rpm for 10 min at 4°C to separate serum.Separated serum was aliquoted and frozen at -80°C for subsequent use in ELISA, ADCD and neutralization assays.Spleens were processed into single-cell suspensions and resuspended in RPMI medium supplemented with 1% penicillin/streptomycin and 10% FBS.Cell concentrations and viabilities were determined using a Countess Automated Cell Counter (Invitrogen, Life Technologies).

ELISA assays
ELISA was used to determine SOSIP-speci c IgG, IgG1, IgG2a, IgG2b and IgG3 present in mouse serum.Mouse blood samples were collected via cheek bleed or cardiac puncture.Ni-NTA HisSorb plates (Qiagen, Cat:35061) were coated with 100 µl/well of recombinant his-tagged BG505-SOSIP.v8.1 protein diluted in 1xTBS (Sera Care or Fisher) to a concentration of 1-2 µg/ml and incubated at room temperature for 2 hrs.Plates were washed with 1xTBS before the addition of diluted mouse serum.For IgG and IgG1 ELISAs, mouse sera were diluted 1:100 in 1xTBS with 2% non-fat dry milk (Rockland Immunochemicals).For IgG2a, IgG2b and IgG3 ELISAs, mouse sera were diluted 1:25 in 1xTBS with 2% non-fat dry milk.

Area under the curve analysis
For each mouse, area under the curve (AUC) was determined for SOSIP-speci c IgG across the following timepoints: day 14 post 1 immunization, day 21 post 2 immunizations, and day 14 post 3 immunizations.Outliers were determined and removed using the robust regression and outlier removal algorithm in GraphPad Prism.AUC was calculated using the AUC analysis function in GraphPad Prism.Statistical signi cances (p < 0.05) between different experimental group AUC values were determined by nonparametric Mann-Whitney U-test.

ELISpot assay
PVDF membrane ELISpot plates (Mabtech, Cat: 3654WP10) were activated with 35% ethanol and washed with PBS prior to coating.Plates were coated with mouse anti-IFN-γ capture antibody (R&D, Cat: SEL485) and incubated overnight at 4°C.Plates were washed with PBS and blocked for 2 hrs at room temperature with PBS containing 1% BSA and 5% sucrose.Splenocytes from vaccinated mice were added (2x10 5 cells/well) and stimulated with ve BG505 SOSIP-speci c peptide pools at a concentration of 2.5 µg/mL per peptide (Cat: ARP-13123, NIH HIV reagent program).A stimulation with an equal percentage amount of DMSO was performed as a negative control, while PMA/Ionomycin (ebioscience, Cat:00-4970-93) was included as a positive control.Cells were stimulated overnight at 37°C with 5% CO 2 .Plates were then washed and incubated with mouse IFN-γ detection antibody (R&D, Cat: SEL485) overnight at 4°C.Plates were washed and incubated with Streptavidin-ALP antibody for 2 hrs at room temperature prior to development.To develop, plates were incubated with BCIP/NBT substrate for 30 mins at room temperature protected from light.Plates were washed and left to dry overnight at room temperature.Plates were then scanned and counted using CTL ImmunoSpot S5 Core Analyzer (Cellular Technology Limited CTL).

Neutralization assay
Pseudoviruses were produced using the pSG3ΔEnv DNA plasmid encoding the HIV backbone and a plasmid encoding (Q461e2TAIV) envelope as previously described [40].Brie y, 293T cells were plated at 5 × 10 6 cells/ ask in a T-75cm ask in medium (DMEM, 10% fetal calf serum, 1% l-glutamine, 1% penicillinstreptomycin).The following day, 293T cells in DMEM without pen/strep were transfected with 10 µg total DNA/ ask at a 20:1 ratio of backbone-to-Env plasmids in the presence of 20 µL polyethylenimine (jetPEI; Polyplus, Inc.) per ask.Cells were incubated for 48 hrs at 37°C.The virus was harvested by the centrifugation of the supernatant at 1,800 rpm for 10 min at 4°C.Pseudovirus stock was aliquoted and stored at -80°C until use.The pseudovirus was titrated on TZM-bl cells; dilutions of the pseudovirus were used to infect TZM-bl cells in the presence of 7.5 µg/ml DEAE-dextran (Fisher Biotech, Fair Lawn, NJ).Two days later, infection was measured by luciferase expression, and 50% tissue culture infective doses (TCID50) (determined by the Reed-Muench formula) were added to each neutralization assay.
The neutralization assays were performed in TZM-bl cells which have an HIV Trans-activator of Transcription (tat) induced luciferase reporter.A 3-fold dilution of the mouse serum was incubated with the tier 1A virus Q461e2TAIV for 1 hr at 37°C.TZM-bl cells were added with DEAE-Dextran and allowed to incubate for 48 hrs at 37°C with 5% CO 2 .Cells were lysed with Bright-Glo (Promega) for 2 mins before being read in a luminometer.ID 50 's were calculated as a 50% reduction in the relative light units (RLU) in the mouse serum dilution wells as compared to the virus-only wells after subtraction of the cell-only controls.All samples were tested in duplicate, and response curves were t by nonlinear regression.

Antibody dependent cellular cytotoxicity (ADCD) assay
Antibody-dependent complement deposition (ADCD) was assessed as described previously [41].Brie y, biotinylated BG505 protein was coupled to uorescence NeutrAvidin beads (Thermo Fisher Scienti c).Mouse sera antibodies were diluted 1:10 in 0.1% BSA and incubated with the coupled BG505 beads for 2 hrs at 37°C.Beads were washed and incubated with complement factors from guinea pig (Sigma-Aldrich) for 20 mins at 37°C.The complement reaction was then stopped by washing with 15 mM EDTA in PBS.
C3 deposition on the beads was detected with a 1:100 diluted FITC-conjugated anti-guinea pig C3 polyclonal antibody (MP Biomedicals, Cat: 0855385) and relative C3 deposition was analyzed by ow cytometry.

Statistical analysis
All statistics were analyzed using GraphPad Prism 9 or 10.Error bars represent means ± standard deviations.Outliers were determined and removed using the robust regression and outlier removal (ROUT) algorithm.Mann-Whitney U test or Kruskal-Wallis ANOVA statistical tests were performed where denotated to determine statistical differences between groups.In all data, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

ADA-1 enhances HIV speci c IgG and Antibody Dependent Complement Deposition (ADCD)
To evaluate the effect of ADA-1 on HIV speci c humoral responses, 6-8-week-old female BALB/c mice were immunized thrice with 1µg pBG505-TTT or 5 µg pBG505-TTT alone or in combination with 10 µg pADA.Control mice were immunized with 15 µg Pvax, an empty plasmid vector, to ensure mice received equal total amounts of DNA and to control for any nonspeci c DNA-induced innate immune responses.
In addition to ADCD, Ab neutralization capacity was evaluated.Mice immunized 5 µg pBG505-TTT alone (***p = 0.0006, mean ID50 = 156.29)and with pADA (**p = 0.0014, mean ID50 = 127.59)had signi cantly increased neutralization against the tier 1A pseudovirus Q461e2TAIV compared to Pvax mice.However, there was no difference in neutralization capacity between mice that received pBG505 alone or with pADA (Supplemental Fig. 1d).Additionally, we observed no correlation between SOSIP-speci c IgG magnitude and neutralization (Supplemental Fig. 1e).Although this study strictly used female BALBc mice, previous studies using pADA in male and female mice did not show any sex biases nor any species (C57BL/6 or BALBc) biases in humoral or cellular responses [13].Together these data demonstrate that pADA is dose sparing, enhances SOSIP-speci c IgG magnitude and durability as well as modestly increases the ability of SOSIP-speci c antibody to induce ADCD.

ADA-1 enhances HIV speci c antibody isotype switching
To evaluate if ADA-1 enhances isotype switching, we measured SOSIP-speci c IgG1, IgG2a, IgG2b and IgG3 in the sera of immunized mice as shown in Fig. 1a The increase in SOSIP-speci c IgG2a in pADA co-immunized mice was signi cant when compared to mice receiving 1 µg pBG505-TTT alone (*p = 0.0192) and near signi cant when compared to mice receiving 5 µg pBG505-TTT alone (p = 0.0513).These data demonstrate the ability of ADA-1 to uniquely induce SOSIP-speci c IgG2a which is not achieved with pBG505-TTT alone (Fig. 2b).When correlating SOSIP-speci c IgG1 to IgG2a production, we observe a signi cant positive correlation (**p = 0.008) (Fig. 2f).

ADA-1 enhances HIV speci c T cell responses
To quantify HIV-BG505 cell mediated immunity, we performed an IFNγ ELISpot assay using splenocytes from immunized mice as outlined in Fig. 1a.Spleens were harvested D14P3 and stimulated with peptide pools encompassing the HIV BG505 protein.HIV speci c IFNγ production was quanti ed by spot forming units (SFUs) per million splenocytes using an ELISpot assay.In line with the humoral responses (Fig. 1-2), mice co-immunized with pADA and 1 µg pBG505-TTT had signi cantly increased IFNγ SFUs (*p = 0.0455, mean SFU = 1785) compared to mice with 1 µg pBG505-TTT alone (mean SFU = 958).Similar to humoral responses, we observed a plateau in SOSIP-speci c IFNγ production in mice immunized with 5 µg pBG505-TTT alone (mean SFU = 2496) or with pADA (mean SFU = 2312) (Fig. 3a).These data suggest that ADA-1 co-delivery can enhance SOSIP-speci c cellular IFNγ production and humoral responses (Fig. 1) in a dose sparing manner.
To understand immunogenic epitopes driving T cell responses, we colored the BG505 peptide pools onto the corresponding regions of the BG505 protein using a cryo-EM structure of a BG505 SOSIP.664trimer (PDB ID: 6V0R).The peptides included in pool 5 have not been structurally resolved on the BG505 protein and are therefore not included.To determine the CD4 binding site, a cryo-EM structure of gp140 bound to the extracellular domains of CD4 (PDB ID: 7T0O) was aligned to the BG505 SOSIP.664trimer (Fig. 3b-c).T cell responses against pBG505-TTT were primarily driven by the BG505 peptide pool 2 and pool 3. Pool 2 and 3 spanned the majority of the gp120 subunit.Notably, these peptide pools spanned important epitopes of BG505 including variable loops (V1/2, V3, V4 and V5), conserved domains (C2, C3, C4 and C5) and the CD4 binding site (Fig. 3b-c).These responses against peptide pool 2 and pool 3 were not necessarily enhanced with ADA-1, however, these ndings demonstrate the ability of pBG505-TTT to drive T cell responses against important epitopes of the BG505 protein.Studies performing T cell epitope mapping of BG505 have been limited but have demonstrated major T cell epitopes within BG505 as C1, C2, C4, C5, V4 in gp120 and HR1 in gp41 [42] which is in line with our ndings presented here.
The gating strategy used is displayed in Supplemental Fig. 2.
Taken together, these data demonstrate that ADA-1 enhances HIV speci c T cell cytokine production and polyfunctionality of effector T cells.These data also suggest that ADA-1 enhances CD4 + and CD8 + effector memory T cell responses but has little to no effect on central memory T cell responses.

ADA-1 enhances HIV speci c T activation memory B cell frequencies
To further quantify HIV-BG505 cell mediated immunity, we performed an activation induced marker (AIM) assay and quanti ed SOSIP-speci c memory B cells using ow cytometry.The AIM assay, as previously reported [44], is used to assess antigen speci c activation of CD4 + T cells through the upregulation of activation markers such as PDL1, OX40 and CD25 upon stimulation.This assay has been described to enrich and monitor antigen speci c TFH cells.For these experiments, splenocytes from immunized mice as outlined in Fig. 1a were stimulated with peptide pools encompassing the HIV BG505 protein and PDL1, OX40 and CD25 expression on CD + T cells was quanti ed via ow cytometry.The gating strategy used is shown in Supplemental Fig. 4.
To evaluate SOSIP-speci c memory B cell responses, we stained splenocytes from immunized mice as outlined in Fig. 1a with uorescently labeled multimerized probes speci c to SOSIP and de ned memory B cells as splenocytes that were CD19 + IgD − via ow cytometry.
Taken together, these data demonstrate that ADA-1 enhances HIV-speci c CD4 + T activation and SOSIPspeci c memory B cells when antigen dose sparing is applied.

ADA-1 induces qualitative differences in the neutralization capacity of HIV-speci c antibodies
We have previously demonstrated that co-immunization with a plasmid-encoded monomeric gp160 HIV envelope DNA vaccine, recombinant gp160 protein, and pADA in mice resulted in enhanced neutralization of the tier 1A pseudovirus MW965 [12].In these studies, enhanced neutralization was only the case upon co-immunization with DNA, protein and pADA and was not the case with DNA alone, DNA and protein or DNA and pADA [12].For this reason, we believed that to see any qualitative difference in Ab responses within a BALB/c mouse model, immunization with DNA, protein and an adjuvant would be necessary.To address this, we immunized mice simultaneously with 10 µg pBG505-TTT alone or with pADA in the right tibialis anterior (TA) muscle and 10 µg rBG505 with alum or with the MF59-like adjuvant AddaVax in the left TA muscle.To understand how pADA performs compared to Alum and AddaVax, we immunized a separate group of mice with 10 µg pBG505-TTT alone or with pADA and no protein co-immunization.As a negative control, we immunized mice with 20µg of Pvax.Mice were immunized thrice and bled on D21P2 and sacri ced at D14P3 (Fig. 6a).
When evaluating SOSIP-speci c IgG, across all immunization groups we observed no difference between mice that received pADA or their non-pADA receiving counterparts (Fig. 6b).This is likely due to the higher dose of pBG505-TTT (10 µg) used for this experiment as we observed a plateau in humoral responses with 5 µg pBG505-TTT (Fig. 1-2).We also evaluated SOSIP-speci c IgA responses and observed no difference between mice that received pADA or their non-pADA receiving counterparts.We did, however, observe a signi cant increase in SOSIP-speci c IgA in mice immunized with pBG505-TTT and pADA (*p = 0.0307) and mice immunized with pBG505, rBG505, Addavax and pADA (*p = 0.0121) compared to Pvax immunized mice.There was an observed trend toward increased SOSIP-speci c IgA in pADA coimmunized mice compared to their non-ADA-1 receiving counterparts although not signi cant (Supplemental Fig. 1c).
When evaluating Ab functionality by pseudoviral neutralization assay, ADA-1 did increase neutralization of the Tier 1A pseudovirus Q461eTAIV to a near signi cant level (p = 0.07) compared to mice immunized with pBG505-TTT, rBG505, and alum.Mice co-immunized with pBG505-TTT, rBG505, and AddaVax had robust neutralization that was not further improved with pADA (Fig. 6c).We observe no correlation between SOSIP-speci c IgG and neutralization ID 50 demonstrating that the increased Ab quality with pADA is not a direct result of increased Ab magnitude (Fig. 6d-f).We did not observe neutralization in any immunization groups against the Tier 1B virus Q23env17 and the autologous BG505 virus (Supplementary Table 1-2) which could in part be due to shortcomings of using wild-type mouse models to evaluate the breadth of antibody neutralization.Collectively these data demonstrate the ability of ADA-1 to enhance the quality of SOSIP-speci c Abs by improving neutralization capacity particularly against Tier 1A pseudovirus Q461eTAIV.

Discussion
We have previously demonstrated that a novel molecular immune modulator, adenosine deaminase-1 (ADA-1) enhances the magnitude and durability of both cellular and humoral vaccine-induced immune responses in vivo [12][13][14].Additionally, we have shown that ADA-1 is critical to the T follicular helper (TFH) program and that the addition of exogenous ADA-1 enhanced the ability of less e cient pre-TFH to provide help to B cells [20].We believe ADA-1 is therefore unique as an immune modulator as it targets TFH cells, and TFH cells are central to many aspects critical for effective anti-HIV humoral and cellular responses.TFH cells are known to be critical orchestrators of germinal center reactions which are needed for long-lived humoral and cellular responses.TFH cells are critical to several processes, such as isotype switching, somatic hypermutation, and a nity maturation in B cells, which in turn are needed for the development of bNAbs and therefore critical for an effective anti-HIV humoral response [45].TFH cells have also been found to boost CD8 + T function and enhanced CD8 + T function has been implicated in enhanced protection against HIV [33][34][35].We therefore hypothesized that ADA-1 combined with pBG505-TTT would result in enhanced magnitude and quality of humoral and cellular HIV speci c responses.Our ndings demonstrate that ADA-1 has a strong impact on both cellular and humoral immune responses particularly CD8 + T cells and qualitative Ab responses which are important contributors to anti-HIV immune responses.
In this study we demonstrated that pADA co-immunization with pBG505-TTT enhances HIV speci c humoral and cellular responses.In terms of humoral responses, we observed that pADA co-immunization increases SOSIP-speci c Ab magnitude, durability, and isotype switching.This is directly in line and supports our previous ndings of pADA enhancing humoral responses to SARS-CoV-2 [13,14] and monomeric gp160 HIV envelope [12] DNA vaccines.ADA-1 co-immunized mice exhibited a modest increase in ADCD suggesting the induction of nNAbs that are mediating this FcγR-mediated Ab function.ADA-1 could therefore be driving ADCD which has been shown to decrease HIV replication and may play a role in HIV protection [25][26][27][28][29][30].
ADA-1 induced robust SOSIP-speci c IgG, IgG1, IgG2a and IgG2b antibodies at strikingly low doses of pBG505-TTT (1µg) and in a dose-sparing manner.Studies using plasmid encoded MD39, another nativelike Env immunogen, required doses of 25µg of DNA to see improved humoral responses [11].In this study, we found that pADA uniquely induces IgG2a production which was not achieved with pBG505-TTT alone.This is in line with our previous ndings of pADA driving TH1-type humoral responses and gene expression pro les [14].IgG2a production is driven by TH1 responses and, functionally, IgG2a has been correlated with in uenza viral clearance and protection against challenge in mice [46] as well as increased overall e cacy of in uenza vaccination [47][48][49][50], suggesting an important role of IgG2a in viral infections.When considering a nity for Fc receptors to mediate antibody dependent effector functions, mouse IgG2a seems to be a functional analog to human IgG3 [51][52][53].HIV infected elite controllers have enhanced antibody functionality including ADCC, monocyte and neutrophil phagocytosis and ADCD primarily coordinated through human IgG3/IgG1 responses [54].Therefore, the ADA-1 driven enhancement of mouse IgG2a observed could be contributing to enhanced ADCD observed here and suggests ADA-1 could induce human IgG3 with enhanced functionality similar to that observed in elite controllers.Additionally, we found that ADA-1 induced a SOSIP-speci c IgA response when co-immunized with pBG505, rBG505, and Addavax suggesting ADA-1 can induce mucosal immunity critical for protection against HIV infection.
This study also demonstrated the ability of ADA-1 to enhance antibody quality.We observed that pADA co-immunization enhanced SOSIP-speci c Ab neutralization compared to mice immunized with pBG505, rBG505 and alum.Increased neutralization capacity is presumably a result of increased somatic hypermutation and antibody a nity.This supports our previous ndings of increased antibody neutralization and a nity with pADA co-immunization [12][13][14].Collectively these ndings suggest that ADA-1 is driving key humoral responses that would be necessary for an effective HIV vaccine.
In terms of cellular responses, we demonstrated in this study that ADA-1 enhances HIV-speci c CD4 + and CD8 + T cell responses.ADA-1 co-immunization led to increased HIV-speci c IFN-γ production in splenocytes, presumably from both CD4 + and CD8 + T cells.T cell responses against pBG505-TTT were primarily driven by the BG505 peptide pool 2, which encompassed the variable loop V3 and CD4 binding site.ADA-1 co-immunization also led to increased HIV speci c polyfunctionality from both CD4 + and CD8 + T cells as well as enhanced activation in CD4 + T cells and enhanced degranulation (i.e.CD107a expression) in CD8 + T cells.We also demonstrated that ADA-1 enhanced frequencies of CD8 + effector memory T cells.HIV elite controllers have been found to have CD8 + T cells with increased memory and effector potential which importantly provides an increased ability to kill HIV infected cells before progeny virions can be produced [34,[55][56][57].Vaccine strategies designed to enhance CD8 + T cell frequency and effector function have also demonstrated protective HIV immunity in NHP [35].Therefore, the increased CD8 + effector memory T cells induced by ADA-1 could contribute protective HIV immunity.In terms of B cell responses, we also demonstrate the ability of ADA-1 to enhance SOSIP-speci c memory B cell responses.These ndings suggest that ADA-1 is driving key cellular responses that would be necessary for an effective HIV vaccine.
A limitation of this study is the shortcoming of using wild-type mouse models to evaluate the breadth of antibody neutralization.Previous studies have shown the di culty of murine B cells to recognize HIV envelope epitopes necessary for the induction of Tier The current study nonetheless demonstrated an important proof of concept that ADA-1 can act as an immune modulator to enhance HIV-speci c humoral and cellular responses in the context of a BG505-SOSIP DNA vaccine.Notably, enhanced humoral and cellular responses with ADA-1 were achieved at very low doses of pBG505-TTT, exemplifying the ability of ADA-1 to be dose-sparing.This study in addition to our previous ndings [12][13][14]20] demonstrated the ability of ADA-1, when formulated with DNA antigens, to target TFH cells and induce durable, higher-quality antibodies which are critical in the development of protective bNAbs against HIV.Importantly, ADA-1 as an enzyme replacement therapy to treat ADA-SCID has already been approved, is clinically well-tolerated and has a demonstrated safety pro le that could facilitate ADA-1 to be fast-tracked for clinical applications [16].Additionally, BG505-SOSIP is currently being evaluated in several on-going clinical trials (NCT03699241, NCT04177355, NCT05863585, NCT05983874) demonstrating it as a promising vaccine antigen to which responses could be further enhanced with an immune modulator such as ADA-1.

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Figure 6 ADA- 1
Figure 6 [9,59,ralizing antibodies[8].While studies in cows have shown BG505 SOSIP immunization to drive broad and potent HIV-speci c neutralizing antibody[58], the eld has yet to achieve a vaccine that induces bNAb responses in other animal models or in humans[9,59, 60].We demonstrated neutralization against the Tier 1A virus Q461e2TAIV; however, we were unable to achieve neutralization in any immunization groups against the Tier 1B virus Q23env17 and or the autologous BG505 virus.Future investigation is needed in other animal models, such as human immunoglobulin, bNAb knock-in mice [61-65], or NHP to evaluate the extent to which ADA-1 can induce bNAb's for HIV-1.
R.W.S, and G.M.C conceived the project.G.M.C. designed experiments; performed immunizations, bleeds, harvests, and immunological assays; coordinated ADCD and neutralization experiments; and analyzed data.D.J. and E.K. performed immunizations, bleeds, harvests, immunological assays and analyzed data.G.A.C. and I.M.C. were involved in study design, data analysis and interpretation, as well as manuscript editing.P.B. and A.J.H. performed neutralization experiments.M.G. and Y.C.B. performed ADCD experiments.I.D.M.S. designed and validated pBG505-TTT used for