Previously, a designed ankyrin repeat protein, AnkGAG1D4, was generated for intracellular targeting of the HIV-1 capsid domain. The efficiency was satisfactory in interfering with the HIV assembly process. Consequently, improved AnkGAG1D4 binding affinity was introduced by substituting tyrosine (Y) for serine (S) at position 45. However, the intracellular anti-HIV-1 activity of AnkGAG1D4-S45Y has not yet been validated. In this study, the performance of AnkGAG1D4 and AnkGAG1D4-S45Y in inhibiting wild-type HIV-1 and HIV-1 maturation inhibitor-resistant replication in SupT1 cells was evaluated. HIV-1 p24 and viral load assays were used to verify the biological activity of AnkGAG1D4 and AnkGAG1D4-S45Y as assembly inhibitors. In addition, retardation of syncytium formation in infected SupT1 cells was observed. Of note, the defense mechanism of both ankyrins did not induce the mutation of target amino acids in the capsid domain. The present data show that the potency of AnkGAG1D4-S45Y was superior to AnkGAG1D4 in interrupting either HIV-1 wild-type or the HIV maturation inhibitor-resistant strain.
Adult-onset immunodeficiency syndrome (AOID) patients with autoantibodies (autoAbs) against interferon-gamma (IFN-γ) generally suffer from recurrent and recalcitrant disseminated non-tuberculous mycobacterial diseases. Since the early stages of AOID do not present specific symptoms, diagnosis and treatment of the condition are not practical. A simplified diagnostic method for differentiating AOID from other immunodeficiencies, such as HIV infection, was created. Anti-IFN-γ is generally identified using enzyme-linked immunosorbent assay (ELISA), which involves an instrument and a cumbersome process. Recombinant IFN-γ indirectly conjugated to colloidal gold was used in the modified immunochromatographic (IC) strips. The biotinylated-IFN-γ was incorporated with colloidal-gold-labeled 6HIS-maltose binding protein-monomeric streptavidin (6HISMBP-mSA) and absorbed at the conjugate pad. The efficacy of the IC strip upon applying an anti-IFN-γ autoAb cut-off ELISA titer of 2500, the sensitivity and specificity were 84% and 90.24%, respectively. When a cut-off ELISA titer of 500 was applied, the sensitivity and specificity were 73.52% and 100%, respectively.
Persistent and efficient therapeutic protein expression in the specific target cell is a significant concern in gene therapy. The controllable integration site, suitable promoter, and proper codon usage influence the effectiveness of the therapeutic outcome. Previously, we developed a non-immunoglobulin scaffold, alpha repeat protein (αRep4E3), as an HIV-1 RNA packaging interference system in SupT1 cells using the lentiviral gene transfer. Although the success of anti-HIV-1 activity was evidenced, the integration site is uncontrollable and may not be practical for clinical translation. In this study, we use the CRISPR/Cas9 gene editing technology to precisely knock-in αRep4E3 genes into the adeno-associated virus integration site 1 (AAVS1) safe harbor locus of the target cells. We compare the αRep4E3 expression under the regulation of three different promoters, including cytomegalovirus (CMV), human elongation factor-1 alpha (EF1α), and ubiquitin C (UbC) promoters with and without codon optimization in HEK293T cells. The results demonstrated that the EF1α promoter with codon-optimized αRep4E3mCherry showed higher protein expression than other promoters with non-optimized codons. We then performed a proof-of-concept study by knocking in the αRep4E3mCherry gene at the AAVS1 locus of the Jurkat cells. The results showed that the αRep4E3mCherry-expressing Jurkat cells exhibited anti-HIV-1 activities against HIV-1NL4-3 strain as evidenced by decreased capsid (p24) protein levels and viral genome copies as compared to the untransfected Jurkat control cells. Altogether, our study demonstrates that the αRep4E3 could interfere with the viral RNA packaging and suggests that the αRep4E3 scaffold protein could be a promising anti-viral molecule that offers a functional cure for people living with HIV-1.
CD147 is upregulated in cancers, including aggressive T-ALL. Traditional treatments for T-ALL often entail severe side effects and the risk of relapse, highlighting the need for more efficacious therapies. ADCP contributes to the antitumor response by enhancing the ability of phagocytic cells to engulf cancer cells upon antibody binding. We aimed to engineer CD147
Current quantitative gene expression detection in genomic and transcriptomic research heavily relies on quantitative real-time PCR (qPCR). While existing multiplex gene detection techniques offer simultaneous analysis of multiple targets, we present an alternative assay capable of detecting gene expression simultaneously within a single well. This highly sensitive method utilizes πCode MicroDiscs, featuring unique identification patterns and fluorescent detection. Our study compared this multiplex πCode platform with a qPCR platform for profiling cytokine gene expression. The πCode MicroDisc assay successfully demonstrated the expression of polymerization markers for M1- and M2-like macrophages generated from THP-1-derived macrophages in a qualitative assay. Additionally, our findings suggest a pattern agreement between the πCode assay and the qPCR assay, indicating the potential of the πCode technology for comparative gene expression analysis. Regarding the inherent sensitivity and linearity, the developed πCode assay primarily provides qualitative gene expression to discriminate the polarization of macrophages. This remarkable capability presents substantial advantages for researchers, rendering the technology highly suitable for high-throughput applications in clinical diagnosis and disease monitoring.
A designed repeat scaffold protein (AnkGAG1D4) recognizing the human immunodeficiency virus-1 (HIV-1) capsid (CA) was formerly established with antiviral assembly. Here, we investigated the molecular mechanism of AnkGAG1D4 function during the late stages of the HIV-1 replication cycle. By applying stimulated emission-depletion (STED) microscopy, Gag polymerisation was interrupted at the plasma membrane. Disturbance of Gag polymerisation triggered Gag accumulation inside producer cells and trapping of the CD81 tetraspanin on the plasma membrane. Moreover, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) experiments were performed to validate the packaging efficiency of RNAs. Our results advocated that AnkGAG1D4 interfered with the Gag precursor protein from selecting HIV-1 and cellular RNAs for encapsidation into viral particles. These findings convey additional information on the antiviral activity of AnkGAG1D4 at late stages of the HIV-1 life cycle, which is potential for an alternative anti-HIV molecule.
Several anti-HIV scaffolds have been proposed as complementary treatments to highly active antiretroviral therapy. AnkGAG1D4, a designed ankyrin repeat protein, formerly demonstrated anti-HIV-1 replication by interfering with HIV-1 Gag polymerization. However, the improvement of the effectiveness was considered. Recently, the dimeric molecules of AnkGAG1D4 were accomplished in enhancing the binding activity against HIV-1 capsid (CAp24). In this study, the interaction of CAp24 against the dimer conformations was elucidated to elaborate the bifunctional property. The accessibility of the ankyrin binding domains was inspected by bio-layer interferometry. By inverting the second module of dimeric ankyrin (AnkGAG1D4NC-CN), the CAp24 interaction KD was significantly reduced. This reflects the capability of AnkGAG1D4NC-CN in simultaneously capturing CAp24. On the contrary, the binding activity of dimeric AnkGAG1D4NC-NC was indistinguishable from the monomeric AnkGAG1D4. The bifunctional property of AnkGAG1D4NC-CN was subsequently confirmed in the secondary reaction with additional p17p24. This data correlates with the MD simulation, which suggested the flexibility of the AnkGAG1D4NC-CN structure. The CAp24 capturing capacity was influenced by the distance of the AnkGAG1D4 binding domains to introduce the avidity mode of AnkGAG1D4NC-CN. Consequently, AnkGAG1D4NC-CN showed superior potency in interfering with HIV-1 NL4-3 WT and HIV-1 NL4-3 MIRCAI201V replication than AnkGAG1D4NC-NC and an affinity improved AnkGAG1D4-S45Y.
BioPhi-guided humanization was utilized to enhance the humanness of a humanized single-chain variable fragment targeting CD99, leading to the development of two variants: HuScFvMT99/3BP and HuScFvMT99/3HY. The HuScFvMT99/3BP variant incorporated framework region modifications, leading to modest improvements in humanness, particularly in the VH domain, although the VL domain remained suboptimal. To address this limitation, HuScFvMT99/3HY was designed by combining the VL domain of wild-type with the VH domain of HuScFvMT99/3BP. Molecular dynamics simulations employing AlphaFold2, AlphaFold3, and HADDOCK were performed to evaluate the HuScFv-CD99 peptide complexes. AF2-based simulations demonstrated enhanced binding free energy (ΔGbinding) for both variants compared to HuScFvMT99/3WT. However, ΔGbinding values obtained from AF3 and HD simulations were inconsistent, with HuScFvMT99/3BP exhibiting the weakest binding affinity. While ΔGbinding patterns derived from AlphaFold3 and HADDOCK simulations aligned, amino acid decomposition analysis revealed variations in the interaction coordinates of the predicted complexes. Root-mean-square deviation analysis indicated improved structural stability for HuScFvMT99/3BP (0.975 Å) and HuScFvMT99/3HY (1.075 Å) relative to HuScFvMT99/3WT (1.225 Å). Biolayer interferometry further confirmed that HuScFvMT99/3WT exhibited the highest binding affinity (KD = 1.35 × 10⁻⁷ M) compared to HuScFvMT99/3BP (KD = 2.64 × 10⁻⁷ M) and HuScFvMT99/3HY (KD = 3.95 × 10⁻⁷ M). Supporting evidence was provided by ELISA and flow cytometry experiments. PITHA analysis revealed a high immunogenicity risk for all variants, despite HuScFvMT99/3HY displaying improved humanness, a larger complementarity-determining region (CDR) cavity, and a more hydrophobic CDR-H3 loop. These findings highlight the delicate balance between enhancing humanness and preserving the structural and functional integrity critical for therapeutic antibody development.
Protein and DNA interactions are crucial for many cellular processes. Biolayer Interferometry (BLI) is a label-free technology for determining kinetic biomolecular interactions with high accuracy results. In the present study, we determined the kinetic binding of a zinc finger scaffold, 2LTRZFP, which formerly constructed the interfering effect on HIV-1 integration process using BLI. The competitive Enzyme-linked immunosorbent assay (ELISA) was used to initially show the specific binding of 2LTRZFP. The percentages of inhibition were 62% and 22% in double-stranded 2LTR (ds2LTR) and irrelevant DNA (dsNeg), respectively. Consequently, the binding affinity of 2LTRZFP against ds2LTR target analyzed by BLI was 40 nM, which is stronger than the interaction of HIV-1 integrase (IN) enzyme to the 2LTR circle junction. Additionally, the 2LTRZFP did not interact with the genomic DNA extracted from SupT1 cell line. This result indicates that 2LTRZFP did not exhibit off-target effects against human genome. The knowledge obtained from this study supports the prospect of using 2LTRZFP in HIV-1 gene therapy.
