Yin Yang 1 (YY1) is a multifunctional zinc finger transcription factor that regulates many key cellular processes. In this study, we report the cloning of YY1 from Litopenaeus vannamei shrimp (LvYY1). This study shows that LvYY1 is ubiquitously expressed in shrimp tissues, and knockdown of LvYY1 expression by doublestranded RNA (dsRNA) injection in white spot syndrome virus (WSSV)-infected shrimp reduced both mRNA levels of the WSSV immediate early gene ie1 as well as overall copy numbers of the WSSV genome. The cumulative mortality rate of infected shrimp also declined with LvYY1 dsRNA injection. Using an insect cell model, we observed that LvYY1 activates ie1 expression, and a mutation introduced into the ie1 promoter subsequently repressed this capability. Moreover, reporter assay results suggested that LvYY1 is involved in basal transcriptional regulation via an interaction with L. vannamei TATA-binding protein (LvTBP). Electrophoretic mobility shift assay (EMSA) results further indicated that LvYY1 binds to a YY1-binding site in the region between positions Ϫ119 and Ϫ126 in the ie1 promoter. Chromatin immunoprecipitation analysis also confirmed that LvYY1 binds to the ie1 promoter in WSSVinfected shrimp. Taken together, these results indicate that WSSV uses host LvYY1 to enhance ie1 expression via a YY1-binding site and the TATA box in the ie1 promoter, thereby facilitating lytic activation and viral replication. IMPORTANCE WSSV has long been a scourge of the shrimp industry and remains a serious global threat. Thus, there is a pressing need to understand how the interactions between WSSV and its host drive infection, lytic development, pathogenesis, and mortality. Our successful cloning of L. vannamei YY1 (LvYY1) led to the elucidation of a critical virus-host interaction between LvYY1 and the WSSV immediate early gene ie1. We observed that LvYY1 regulates ie1 expression via a consensus YY1-binding site and TATA box. LvYY1 was also found to interact with L. vannamei TATAbinding protein (LvTBP), which may have an effect on basal transcription. Knockdown of LvYY1 expression inhibited ie1 transcription and subsequently reduced viral DNA replication and decreased cumulative mortality rates of WSSV-infected shrimp. These findings are expected to contribute to future studies involving WSSV-host interactions.

The highly conserved matrix protein 2 ectodomain (M2e) of influenza viruses presents a compelling vaccine antigen candidate for stemming the pandemic threat of the mutation-prone pathogen, yet the low immunogenicity of the diminutive M2e peptide renders vaccine development challenging. A highly potent M2e nanoshell vaccine that confers broad and durable influenza protectivity under a single vaccination is shown. Prepared via asymmetric ionic stabilization for nanoscopic curvature formation, polymeric nanoshells co-encapsulating high densities of M2e peptides and stimulator of interferon genes (STING) agonists are prepared. Robust and long-lasting protectivity against heterotypic influenza viruses is achieved with a single administration of the M2e nanoshells in mice. Mechanistically, molecular adjuvancy by the STING agonist and nanoshell-mediated prolongation of M2e antigen exposure in the lymph node follicles synergistically contribute to the heightened anti-M2e humoral responses. STING agonist-triggered T cell helper functions and extended residence of M2e peptides in the follicular dendritic cell network provide a favorable microenvironment that induces Th1-biased antibody production against the diminutive antigen. These findings highlight a versatile nanoparticulate design that leverages innate immune pathways for enhancing the immunogenicity of weak immunogens. The single-shot nanovaccine further provides a translationally viable platform for pandemic preparedness.

The recent Zika virus (ZIKV) epidemic poses a serious threat to global health due to its association with microcephaly and congenital diseases in newborns and neurological complications and Guillain-Barré syndrome in adults. However, the majority of people infected with ZIKV do not develop symptoms. The platforms aimed to specifically diagnose ZIKV infection are needed for patient care and public health surveillance. In the study, four ZIKV envelope (E) protein-specific monoclonal antibodies (mAbs) (A1, B1, C1, and 9E-1) have been developed by using the conventional mAb technology. The binding epitopes of mAbs A1, B1, C1, and 9E-1 are located at E(238-257), E(410-431), E(258-277), and E(340-356), respectively. mAb 9E-1 performs 1.4to 47-fold strong affinity to ZIKV E protein compared to another three mAbs. mAbs A1, C1, and 9E-1 do not have crossreactivity against the recombinant E proteins of dengue virus serotypes 2, 3, and 4. Although these four mAbs do not have ZIKV neutralizing activity, mAbs B1 and 9E-1 have been developed as the lateral flow immunochromatographic assay for specific detection of ZIKV E protein and virions. Key points• The mAbs targeting to the regions of E(238-257), E(410-431), E(258-277), and E(340-356) do not have ZIKV neutralizing activity.• The binding epitope of mAb 9E-1 is highly specific to ZIKV E protein.• mAbs B1 and 9E-1 can bind to ZIKV virions and have been developed as the lateral flow immunochromatographic assay.

Recombinant proteins are essential in the development of subunit vaccines. In the design of many recombinant proteins, polyhistidine residues are added to the N- or C-termini of target sequences to facilitate purification. However, whether the addition of tag residues influences the immunogenicity of proteins remains unknown. In this study, the tag-free SARS-CoV-2 RBD and His-tag SARS-CoV-2 RBD proteins were investigated to determine whether there were any differences in their receptor binding affinity and immunogenicity. The results showed that the tag-free RBD protein had a higher affinity for binding with hACE2 receptors than His-tag RBD proteins (EC50: 1.78 µM vs. 7.51 µM). On day 21 after primary immunization with the proteins, the serum ELISA titers of immunized mice were measured and found to be 1:1418 for those immunized with tag-free RBD and only 1:2.4 for His-tag RBD. Two weeks after the booster dose, tag-free-RBD-immunized mice demonstrated a significantly higher neutralizing titer of 1:369 compared with 1:7.9 for His-tag-RBD-immunized mice. Furthermore, neutralizing antibodies induced by tag-free RBD persisted for up to 5 months and demonstrated greater cross-neutralization of the SARS-CoV-2 Delta variant. Evidence from Western blotting showed that the serum of His-tag-RBD-immunized mice recognized irrelevant His-tag proteins. Collectively, we conclude that the addition of a polyhistidine tag on a recombinant protein, when used as a COVID-19 vaccine antigen, may significantly impair protein immunogenicity against SARS-CoV-2. Antibody responses induced were clearly more rapid and robust for the tag-free SARS-CoV-2 RBD than the His-tag SARS-CoV-2 RBD. These findings provide important information for the design of antigens used in the development of COVID-19 subunit vaccines.

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