Protein-protein interaction plays an essential role in almost all cellular processes and biological functions. Coupling molecular dynamics (MD) simulations and nanoparticle tracking analysis (NTA) assay offered a simple, rapid, and direct approach in monitoring the protein-protein binding process and predicting the binding affinity. Our case study of designed ankyrin repeats proteins (DARPins)—AnkGAG1D4 and the single point mutated AnkGAG1D4-Y56A for HIV-1 capsid protein (CA) were investigated. As reported, AnkGAG1D4 bound with CA for inhibitory activity; however, it lost its inhibitory strength when tyrosine at residue 56 AnkGAG1D4, the most key residue was replaced by alanine (AnkGAG1D4-Y56A). Through NTA, the binding of DARPins and CA was measured by monitoring the increment of the hydrodynamic radius of the AnkGAG1D4-gold conjugated nanoparticles (AnkGAG1D4-GNP) and AnkGAG1D4-Y56A-GNP upon interaction with CA in buffer solution. The size of the AnkGAG1D4-GNP increased when it interacted with CA but not AnkGAG1D4-Y56A-GNP. In addition, a much higher binding free energy (∆GB) of AnkGAG1D4-Y56A (−31 kcal/mol) obtained from MD further suggested affinity for CA completely reduced compared to AnkGAG1D4 (−60 kcal/mol). The possible mechanism of the protein-protein binding was explored in detail by decomposing the binding free energy for crucial residues identification and hydrogen bond analysis.

Concerns over vaccine efficacy after the emergence of the SARS-CoV-2 Delta variant prompted revisiting the vaccine design concepts. Monoclonal antibodies (mAbs) have been developed to identify the neutralizing epitopes on spike protein. It has been confirmed that the key amino acid residues in epitopes that induce the formation of neutralizing antibodies do not have to be on the receptor-binding domain (RBD)- angiotensin-converting enzyme 2 (ACE2) contact surface, and may be conformationally hidden. In addition, this epitope is tolerant to amino acid mutations of the Delta variant. The antibody titers against RBD in health care workers in Thailand receiving two doses of CoronaVac, followed by a booster dose of BNT162b2, were significantly increased. The neutralizing antibodies against the Delta variant suggest that the overall neutralizing antibody level against the Wuhan strain, using the NeutraLISA, was consistent with the levels of anti-RBD antibodies. However, individuals with moderate anti-RBD antibody responses have different levels of a unique antibody population competing with a cross-neutralizing mAb clone, 40591-MM43, determined by in-house competitive ELISA. Since 40591-MM43 mAb indicates cross-neutralizing activity against the Delta variant, this evidence implies that the efficiency of the vaccination regimen should be improved to facilitate cross-protective antibodies against Delta variant infections. The RBD epitope recognized by 40591-MM43 mAb is hidden in the close state.

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.

The diagnosis of mycobacterial infections, including both the Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM), poses a significant global medical challenge. This study proposes a novel approach using immunochromatographic (IC) strip tests for the simultaneous detection of MTBC and NTM. Traditional methods for identifying mycobacteria, such as culture techniques, are hindered by delays in distinguishing between MTBC and NTM, which can affect patient care and disease control. Molecular methods, while sensitive, are resource-intensive and unable to differentiate between live and dead bacteria. In this research, we developed unique monoclonal antibodies (mAbs) against Ag85B, a mycobacterial secretory protein, and successfully implemented IC strip tests named 8B and 9B. These strips demonstrated high concordance rates with conventional methods for detecting MTBC, with positivity rates of 93.9% and 85.9%, respectively. For NTM detection, the IC strip tests achieved a 63.2% detection rate compared to culture methods, considering variations in growth rates among different NTM species. Furthermore, this study highlights a significant finding regarding the potential of MPT64 and Ag85B proteins as markers for MTBC detection. In conclusion, our breakthrough method enables rapid and accurate detection of both MTBC and NTM bacteria within the BACTEC MGIT system. This approach represents a valuable tool in clinical settings for distinguishing between MTBC and NTM infections, thereby enhancing the management and control of mycobacterial diseases. Key points: • Panel of mAbs for differentiating MTB versus NTM • IC strips for diagnosing MTBC and NTM after the BACTEC MGIT • Combined detection of MTP64 and Ag85B enhances diagnostic accuracy

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: HuScFvMT99/3^BP and HuScFvMT99/3^HY. The HuScFvMT99/3^BP 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, HuScFvMT99/3^HY was designed by combining the VL domain of wild-type with the VH domain of HuScFvMT99/3^BP. 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 (ΔG_binding) for both variants compared to HuScFvMT99/3^WT. However, ΔG_binding values obtained from AF3 and HD simulations were inconsistent, with HuScFvMT99/3^BP exhibiting the weakest binding affinity. While ΔG_binding 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 HuScFvMT99/3^BP (0.975 Å) and HuScFvMT99/3^HY (1.075 Å) relative to HuScFvMT99/3^WT (1.225 Å). Biolayer interferometry further confirmed that HuScFvMT99/3^WT exhibited the highest binding affinity (K_D = 1.35 × 10⁻⁷ M) compared to HuScFvMT99/3^BP (K_D = 2.64 × 10⁻⁷ M) and HuScFvMT99/3^HY (K_D = 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 HuScFvMT99/3^HY 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.

Previously, a designed ankyrin repeat protein, Ank^GAG1D4, was generated for intracellular targeting of the HIV-1 capsid domain. The efficiency was satisfactory in interfering with the HIV assembly process. Consequently, improved Ank^GAG1D4 binding affinity was introduced by substituting tyrosine (Y) for serine (S) at position 45. However, the intracellular anti-HIV-1 activity of Ank^GAG1D4-S45Y has not yet been validated. In this study, the performance of Ank^GAG1D4 and Ank^GAG1D4-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 Ank^GAG1D4 and Ank^GAG1D4-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 Ank^GAG1D4-S45Y was superior to Ank^GAG1D4 in interrupting either HIV-1 wild-type or the HIV maturation inhibitor-resistant strain.