B cells have been engineered ex vivo to express an HIV-1 broadly neutralizing antibody (bNAb). B-cell reprograming may be scientifically and therapeutically useful, but current approaches limit B-cell repertoire diversity and disrupt the organization of the heavy-chain locus. A more diverse and physiologic B-cell repertoire targeting a key HIV-1 epitope could facilitate evaluation of vaccines designed to elicit bNAbs, help identity more potent and bioavailable bNAb variants, or directly enhance viral control in vivo. Here we address the challenges of generating such a repertoire by replacing the heavy-chain CDR3 (HCDR3) regions of primary human B cells. To do so, we identified and utilized an uncharacterized Cas12a ortholog that recognizes PAM motifs present in human and murine JH genes. We also optimized the design of 200 nucleotide homology-directed repair templates (HDRT) by minimizing the required 3'-5' resection of the HDRT-complementary strand. Using these techniques, we edited primary human B cells to express a hemagglutinin epitope tag and the HCDR3 regions of the bNAbs PG9 and CH01. Those edited with bNAb HCDR3 efficiently bound trimeric HIV-1 antigens, implying they could affinity mature in vivo in response to the same antigens. This approach generates diverse B-cell repertoires recognizing a key HIV-1 neutralizing epitope.

Background:Highly pathogenic avian influenza H5N1 virus is a serious threat to humans. Due to its antiviral activity, antibody-based therapy is one of the possible effective countermeasures. Here,a combination of intracellular and extracellular human antibodies was investigated and showed a better protective effect. Methods: The scFv4F5-based intracellular antibody and full-length IgG1 extracellular antibody vectors were constructed or expressed, respectively. In vitro, the sensitivity, specificity and affinity of these antibodies were detected by western blotting, ELISA, flow cytometry, Biacore X100 SPR technique and microneutralization assay. In vivo, the protective effect of the combination of antibodies and the dynamics of viral replication were tested, and the related cytokines and proteins were detected by ELISA, western blotting and qPCR. Results: The intracellular antibody could inhibit H5N1 virus propagation in A549 cells in a dose-dependent manner. The protective effect of IgG1 was good in post-treatment therapy in a mouse model. When the intracellular antibody was pre-transfected in a combination regimen with IgG1, it had a better protective effect than IgG1 alone. The protective effect was primarily accomplished by inducing the secretion of cytokines, i.e., IFN-γ, IL-6, and IL-10, and the expression of apoptosis-related proteins, i.e., Bim and cleaved PARP. Conclusions: This antibody combination technique could be used as an appropriate and powerful alternative to antiviral therapy.

Though the 2009 pandemic H1N1 virus has become a seasonal influenza A virus, continued host adaptation and antigenic drift may limit the effectiveness of current vaccines. Here, we assess viral antigenic change of a 2009 pandemic H1N1 virus (A/Changchun/01/2009) in the absence and presence of immune-mediated selective pressures and further identify the key amino acid substitutions involved into the antigenic change of the 2009 pandemic H1N1 virus. We found that serial passage of the 2009 pandemic H1N1 virus in both naïve and previously immunized mice generated antigenically distinct variants and that serial passage in previously immunized mice generated viruses with lower overall antigenic relatedness to the parental virus when compared to variants generated by serial passage in naïve mice, suggesting that antibody pressure may accelerate the antigenic variation rate of the 2009 pandemic H1N1 virus. Furthermore, we found that three amino acid substitutions in the viral HA protein (N159D, S186P, and D225G), both alone and in combination, affected viral antigenicity. It is noted that all three mutations (N159D, S186P, and D225G) in the viral HA protein have been found in the natural 2009 pandemic H1N1 virus isolates and can also enhance the pathogenicity of the 2009 pandemic H1N1 virus to mammals, suggesting that these mutations in the viral HA protein may pose a potential threat to public health and should be paid more attention. Taken together, our work defines some novel molecular determinants of the pandemic H1N1 virus antigenicity and has important implications for ongoing human influenza virus surveillance efforts.