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APOBEC3G (apolipoprotein B mRNA editing enzyme catalytic polypeptide‐like 3G; A3G) is an innate defense protein showing activity against retroviruses and retrotransposons. Activated CD4+ T cells are highly permissive for HIV‐1 replication, whereas resting CD4+ T cells are refractory. Dendritic cells (DCs), especially mature DCs, are also refractory. We investigated whether these differences could be related to a differential A3G expression and/or subcellular distribution. We found that A3G mRNA and protein expression is very low in resting CD4+ T cells and immature DCs, but increases strongly following T‐cell activation and DC maturation. The Apo‐7 anti‐A3G monoclonal antibody (mAb), which was specifically developed, confirmed these differences at the protein level and disclosed that A3G is mainly cytoplasmic in resting CD4+ T cells and immature DCs. Nevertheless, A3G translocates to the nucleus in activated‐proliferating CD4+ T cells, yet remaining cytoplasmic in matured DCs, a finding confirmed by immunoblotting analysis of cytoplasmic and nuclear fractions. Apo‐7 mAb was able to immunoprecipitate endogenous A3G allowing to detect complexes with numerous proteins in activated‐proliferating but not in resting CD4+ T cells. The results show for the first time the nuclear translocation of A3G in activated‐proliferating CD4+ T cells. [ABSTRACT FROM AUTHOR]

Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs)1,2. However even the best bNAbs neutralize 10–50% of HIV-1 isolates inefficiently (IC80 > 5 μg/ml), suggesting that high concentrations of these antibodies would be necessary to achieve general protection3–6. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean IC50 < 0.05 μg/ml). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2, and SIV isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46, and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17 to 77 μg/ml of fully functional rhesus eCD4-Ig for 40 weeks, and these macaques were protected from multiple infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.

Prescreening of participants in clinical trials that use adeno-associated virus (AAV) vectors is required to identify naive participants, as preexisting neutralizing antibodies can limit the efficacy of AAV gene therapies. The presence of antibodies to individual AAV serotypes is typically detected by neutralization assay. To streamline the screening process, we compared an ELISA-based screening method with a neutralization assay for the detection of antibodies against AAV1, AAV8, and AAV9 in a collection of 50 rhesus macaque sera and 20 human sera. We observed a high level of concordance between the two assays (Pearson r > 0.8) for all three serotypes in both sample sets. We thus investigated pre- vs post-vector inoculation sera samples from rhesus macaques that received AAV1 or AAV8 vector inoculations for cross-reactive anti-AAV antibodies. All 12 macaques seroconverted to the vector they received, but many also reacted to the other serotypes. Our results validate an easy-to-use ELISA for reliable detection of antibodies to individual serotypes of AAV. Our results also demonstrate that an antibody response post-AAV inoculation may partially cross-react with other AAV serotypes. Overall, these results suggest that either assay can be used by academic labs for prescreening samples for preexisting anti-AAV antibodies., Competing Interests: M.R.G. is a co-founder and consultant for Emmune, Inc. M.R.G. is an inventor on patents with potential royalties licensed to Emmune, Inc. G.G. is a co-founder of Voyager Therapeutics and Aspa Therapeutics and holds equity in both companies. G.G. is an inventor on patents with potential royalties licensed to Voyager Therapeutics, Aspa Therapeutics, and other biopharmaceutical companies. The remaining authors declare no competing interests., (© 2022 The Authors.)

The absence of fucose on asparagine-297 of the human immunoglobulin G (IgG) heavy chain has been shown to enhance antibody-dependent cellular cytotoxicity (ADCC) activity by 10- to 100-fold compared to fucosylated antibody. Our lab is studying the use of adeno-associated virus (AAV) as a vector for the delivery of HIV-specific antibodies for therapeutic purposes. Since the antibody is produced by vector-transduced cells in vivo , current techniques of glycoengineering cannot be utilized. In order to achieve similar enhancement of ADCC with AAV-delivered antibodies, short hairpin RNAs (shRNAs) that target fucosyltransferase-8 (FUT8), were designed, tested, and cloned into AAV vectors used to deliver HIV-specific broadly neutralizing antibodies (bNAbs). Antibodies produced by our glycoengineered-AAV (GE-AAV) vectors were analyzed for fucose content and ADCC. GE-AAV constructs were able to achieve over 80% knockdown of FUT8. Results were confirmed by lectin western blot for α1-6 fucose, which revealed almost a complete absence of fucose on GE-AAV-produced antibodies. GE-AAV-produced antibodies revealed >10-fold enhancement of ADCC, while showing identical neutralization and gp140 trimer binding compared to their fucosylated counterparts. ADCC was enhanced 40- to 60-fold when combined with key Fc mutations known to enhance binding to FcγRIIIA. Our findings define a powerful approach for supercharging AAV-delivered anti-HIV antibodies., Competing Interests: G.G. is a scientific co-founder of Voyager Therapeutics and holds equity in the company. G.G. is an inventor on patents with potential royalties licensed to Voyager Therapeutics and other biopharmaceutical companies. G.G. is a scientific co-founder of Aspa Therapeutics and holds equity in the company. G.G. is an inventor on patents with potential royalties licensed to Aspa Therapeutics and other bio-pharmaceutical companies. The remaining authors declare no competing interests., (© 2020 The Authors.)

Structural characterization of the HIV-1 Envelope (Env) glycoprotein has facilitated the development of Env probes to isolate HIV-specific monoclonal antibodies (mAbs). However, preclinical studies have largely evaluated these virus-specific mAbs against chimeric viruses, which do not naturally infect non-human primates, in contrast to the unconstrained simian immunodeficiency virus (SIV)mac239 clone. Given the paucity of native-like reagents for the isolation of SIV-specific B cells, we examined a method to isolate SIVmac239-specific mAbs without using Env probes. We first activated virus-specific B cells by inducing viral replication after the infusion of a CD8β-depleting mAb or withdrawal of antiretroviral therapy in SIVmac239-infected rhesus macaques. Following the rise in viremia, we observed 2- to 4-fold increases in the number of SIVmac239 Env-reactive plasmablasts in circulation. We then sorted these activated B cells and obtained 206 paired Ab sequences. After expressing 122 mAbs, we identified 14 Env-specific mAbs. While these Env-specific mAbs bound to both the SIVmac239 SOSIP.664 trimer and to infected primary rhesus CD4 + T cells, five also neutralized SIVmac316. Unfortunately, none of these mAbs neutralized SIVmac239. Our data show that this method can be used to isolate virus-specific mAbs without antigenic probes by inducing bursts of contemporary replicating viruses in vivo ., (© 2020 The Author(s).)

A number of publications have described the use of adeno-associated virus (AAV) for the delivery of anti-HIV and anti-simian immunodeficiency virus (SIV) monoclonal antibodies (mAbs) to rhesus monkeys. Anti-drug antibodies (ADAs) have been frequently observed, and long-term AAV-mediated delivery has been inconsistent. Here, we investigated different AAV vector strategies and delivery schemes to rhesus monkeys using the rhesus monkey mAb 4L6. We compared 4L6 immunoglobulin G1 (IgG1) delivery using the AAV1 versus the AAV8 serotype with a cytomegalovirus (CMV) promoter and the use of a muscle-specific versus a liver-specific promoter. Long-term expression levels of 4L6 IgG1 following AAV8-mediated gene transfer were comparable to those following AAV1-mediated gene transfer. AAV1-mediated gene transfer, using a muscle-specific promoter, showed robust ADAs and transiently low 4L6 IgG1 levels that ultimately declined to below detectable levels. Intravenous AAV8-mediated gene transfer, using a liver-specific promoter, also resulted in low levels of delivered 4L6 IgG1, but those low levels were maintained in the absence of any detectable ADAs. Booster injections using AAV1-CMV allowed for increased 4L6 IgG1 serum levels in animals that were primed with AAV8 but not with AAV1. Our results suggest that liver-directed expression may help to limit ADAs and that re-administration of AAV of a different serotype can result in successful long-term delivery of an immunogenic antibody., (© 2019 The Author(s).)

APOBEC3G (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G; A3G) is an innate defense protein showing activity against retroviruses and retrotransposons. Activated CD4(+) T cells are highly permissive for HIV-1 replication, whereas resting CD4(+) T cells are refractory. Dendritic cells (DCs), especially mature DCs, are also refractory. We investigated whether these differences could be related to a differential A3G expression and/or subcellular distribution. We found that A3G mRNA and protein expression is very low in resting CD4(+) T cells and immature DCs, but increases strongly following T-cell activation and DC maturation. The Apo-7 anti-A3G monoclonal antibody (mAb), which was specifically developed, confirmed these differences at the protein level and disclosed that A3G is mainly cytoplasmic in resting CD4(+) T cells and immature DCs. Nevertheless, A3G translocates to the nucleus in activated-proliferating CD4(+) T cells, yet remaining cytoplasmic in matured DCs, a finding confirmed by immunoblotting analysis of cytoplasmic and nuclear fractions. Apo-7 mAb was able to immunoprecipitate endogenous A3G allowing to detect complexes with numerous proteins in activated-proliferating but not in resting CD4(+) T cells. The results show for the first time the nuclear translocation of A3G in activated-proliferating CD4(+) T cells.

Regulatory T cells have an important role in immune suppression during HIV-1 infection. As regulatory T cells produce the immunomodulatory molecule adenosine, our aim here was to assess the potential of adenosine removal to revert the suppression of anti-HIV responses exerted by regulatory T cells. The experimental setup consisted of ex vivo cocultures of T and dendritic cells, to which adenosine deaminase, an enzyme that hydrolyzes adenosine, was added. In cells from healthy individuals, adenosine hydrolysis decreased CD4(+)CD25(hi) regulatory T cells. Addition of 5'-N-ethylcarboxamidoadenosine, an adenosine receptor agonist, significantly decreased CD4(+)CD25(lo) cells, confirming a modulatory role of adenosine acting via adenosine receptors. In autologous cocultures of T cells with HIV-1-pulsed dendritic cells, addition of adenosine deaminase led to a significant decrease of HIV-1-induced CD4(+)CD25(hi) forkhead box p3(+) cells and to a significant enhancement of the HIV-1-specific CD4(+) responder T cells. An increase in the effector response was confirmed by the enhanced production of CD4(+) and CD8(+) CD25(-)CD45RO(+) memory cell generation and secretion of Th1 cytokines, including IFN-γ and IL-15 and chemokines MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5. These ex vivo results show, in a physiologically relevant model, that adenosine deaminase is able to enhance HIV-1 effector responses markedly. The possibility to revert regulatory T cell-mediated inhibition of immune responses by use of adenosine deaminase, an enzyme that hydrolyzes adenosine, merits attention for restoring T lymphocyte function in HIV-1 infection., (© Society for Leukocyte Biology.)

By interacting with CD26 on the CD4+ T cell surface and with the AdoR A(₂B) on the DC surface, ADA triggers a costimulatory signal for human T cells. The aim of this study was to know whether ADA-mediated costimulation plays a role in the differentiation of T cells. The results show that irrespective of its enzymatic activity and dependent on TNF-α, IFN-γ, and IL-6 action, ADA enhanced the differentiation of CD4+CD45RA+CD45RO⁻ naïve T cells toward CD4+CD25+CD45RO+ Teffs and CD4+CD45RA⁻CD45RO+ memory T cells. Furthermore, ADA potentiated generation of CD4+CD25(high)Foxp3+ Tregs by a mechanism that seems to be mainly dependent on the enzymatic activity of ADA. Interestingly, an ADA-mediated increase on Teff, memory T cell, and Treg generation occurred, not only in cocultures from healthy individuals but also from HIV-infected patients. These data suggest that ADA is a relevant modulator of CD4+ T cell differentiation, even in cells from immunologically compromised individuals.