Broadly neutralizing antibodies to combat influenza virus infection.

The diversified classification and continuous alteration of influenza viruses underscore for antivirals and vaccines that can counter a broad range of influenza subtypes. Hemagglutinin (HA) and neuraminidase (NA) are two principle viral surface targets for broadly neutralizing antibodies. A series of monoclonal antibodies, targeting HA and NA, have been discovered and characterized with a wide range of neutralizing activity against influenza viruses. Clinical studies have demonstrated the safety and efficacy of some HA stem-targeting antibodies against influenza viruses. Broadly neutralizing antibodies (bnAbs) can serve as both prophylactic and therapeutic agents, as well as play a critical role in identifying antigens and epitopes for the development of universal vaccines. In this review, we described and summarized the latest discoveries and advancements of bnAbs against influenza viruses in both pre- and clinical development. Additionally, we assess whether bnAbs can serve as a viable alternative to vaccination against influenza. Finally, we discussed the rationale behind reverse vaccinology, a structure-guided universal vaccine design strategy that efficiently identifies candidate antigens and conserved epitopes that can be targeted by antibodies.

Codelivery of SARS-CoV-2 Prefusion-Spike Protein with CBLB502 by a Dual-Chambered Ferritin Nanocarrier Potentiates Systemic and Mucosal Immunity.

Thousands of breakthrough infections are confirmed after intramuscular (i.m.) injection of the approved vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two major factors might contribute to breakthrough infections. One is the emergence of mutant variants of SARS-CoV-2, and the other is that i.m. injection has an inefficient ability to activate mucosal immunity in the upper respiratory tract. Here, we devised a dual-chambered nanocarrier that can codeliver the adjuvant CBLB502 with prefusion-spike (pre-S) onto a ferritin nanoparticle. This vaccine enabled enhanced systemic and local mucosal immunity in the upper and lower respiratory tract. Further, codelivery of CBLB502 with pre-S induced a Th1/Th2-balanced immunoglobulin G response. Moreover, the codelivery nanoparticle showed a Th1-biased cellular immune response as the release of splenic INF-γ was significantly heightened while the level of IL-4 was elevated to a moderate extent. In general, the developed dual-chambered nanoparticle can trigger multifaceted immune responses and shows great potential for mucosal vaccine development.

Intratumoral delivery of a novel oncolytic adenovirus encoding human antibody against PD-1 elicits enhanced antitumor efficacy.

To date, diverse combination therapies with immune checkpoint inhibitors (ICIs), particularly oncolytic virotherapy, have demonstrated enhanced therapeutic outcomes in cancer treatment. However, high pre-existing immunity against the widely used adenovirus human serotype 5 (AdHu5) limits its extensive clinical application. In this study, we constructed an innovative oncolytic virus (OV) based on a chimpanzee adenoviral vector with low seropositivity in the human population, named AdC68-spE1A-αPD-1, which endows the parental OV (AdC68-spE1A-ΔE3) with the ability to express full-length anti-human programmed cell death-1 monoclonal antibody (αPD-1). In vitro studies indicated that the AdC68-spE1A-αPD-1 retained parental oncolytic capacity, and αPD-1 was efficiently secreted from the infected tumor cells and bound exclusively to human PD-1 (hPD-1) protein. In vivo, intratumoral treatment with AdC68-spE1A-αPD-1 resulted in significant tumor suppression, prolonged overall survival, and enhanced systemic antitumor memory response in an hPD-1 knockin mouse tumor model. This strategy outperformed the unarmed OV and was comparable with combination therapy with intratumoral injection of AdC68-spE1A-ΔE3 and systemic administration of commercial αPD-1. In summary, AdC68-spE1A-αPD-1 is a cost-effective approach with potential clinical applications. ‬‬‬‬.

Unique binding pattern for a lineage of human antibodies with broad reactivity against influenza A virus.

Most structurally characterized broadly neutralizing antibodies (bnAbs) against influenza A viruses (IAVs) target the conserved conformational epitopes of hemagglutinin (HA). Here, we report a lineage of naturally occurring human antibodies sharing the same germline gene, VH3-48/VK1-12. These antibodies broadly neutralize the major circulating strains of IAV in vitro and in vivo mainly by binding a contiguous epitope of H3N2 HA, but a conformational epitope of H1N1 HA, respectively. Our structural and functional studies of antibody 28-12 revealed that the continuous amino acids in helix A, particularly N49HA2 of H3 HA, are critical to determine the binding feature with 28-12. In contrast, the conformational epitope feature is dependent on the discontinuous segments involving helix A, the fusion peptide, and several HA1 residues within H1N1 HA. We report that this antibody was initially selected by H3 (group 2) viruses and evolved via somatic hypermutation to enhance the reactivity to H3 and acquire cross-neutralization to H1 (group 1) virus. These findings enrich our understanding of different antigenic determinants of heterosubtypic influenza viruses for the recognition of bnAbs and provide a reference for the design of influenza vaccines and more effective antiviral drugs.

Histone Deacetylase Inhibitor SAHA Induces Expression of Fatty Acid-Binding Protein 4 and Inhibits Replication of Human Cytomegalovirus.

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor that shows marked efficacy against many types of cancers and is approved to treat severe metastatic cutaneous T-cell lymphomas. In addition to its anticancer activity, SAHA has significant effects on the growth of many viruses. The effect of SAHA on replication of human cytomegalovirus (HCMV) has not, however, been investigated. Here, we showed that the replication of HCMV was significantly suppressed by treatment with SAHA at concentrations that did not show appreciable cytotoxicity. SAHA reduced transcription and protein levels of HCMV immediate early genes, showing that SAHA acts at an early stage in the viral life-cycle. RNA-sequencing data mining showed that numerous pathways and molecules were affected by SAHA. Interferon-mediated immunity was one of the most relevant pathways in the RNA-sequencing data, and we confirmed that SAHA inhibits HCMV-induced IFN-mediated immune responses using quantitative Real-time PCR (qRT-PCR). Fatty acid-binding protein 4 (FABP4), which plays a role in lipid metabolism, was identified by RNA-sequencing. We found that FABP4 expression was reduced by HCMV infection but increased by treatment with SAHA. We then showed that knockdown of FABP4 partially rescued the effect of SAHA on HCMV replication. Our data suggest that FABP4 contributes to the inhibitory effect of SAHA on HCMV replication.

Development of HBc virus-like particles as modular nanocarrier by intein-mediated trans-splicing.

Hepatitis B virus core protein (HBc) spontaneously assembles as Virus-like particles (VLPs) in Escherichia coli (E. coli) which is extensively used as a nanocarrier to boost antigen immunogenicity. Genetic fusion of cargo protein with HBc occasionally forms inclusion bodies instead of properly assembled VLPs. To this end, we devised HBc VLPs as a modular nanocarrier for antigen delivery by intein-mediated trans-splicing (TS). We introduced split inteinC (intC) to the C-terminus of split HBc N-core to employ intein-mediated TS technology to HBc VLPs. Split HBc with the insertion of intC at N-core C-terminus (designated as HBc N-intC-C) existed in inclusion bodies. Interestingly, introduction of a soluble tag, gb1, to intC C-terminus remarkably improved the solubility of recombinant protein (named HBc N-intC-gb1-C). Moreover, newly designed recombinant spontaneously assembled as VLPs and endowed efficiently coupling two different model antigens onto HBc N-intC-gb1-C VLPs. Furthermore, model antigens delivered by HBc VLPs induced a dramatically enhanced antigen-specific immune responses. Antigen proteins mainly elicited Th2 IgG responses while antigens delivered by HBc VLPs steered Th1/Th2 balanced IgG responses. Taken together, intein-mediated TS was amenable to decorate HBc VLPs with antigens and showed good potential for antigen delivery.

Murine Cytomegalovirus Protein pM49 Interacts with pM95 and Is Critical for Viral Late Gene Expression.

Late gene expression of betaherpesviruses and gammaherpesviruses is tightly controlled by virus-encoded transactivation factors (vTFs). We recently proved that the 6 vTFs of murine cytomegalovirus (MCMV) form a complex to regulate late gene transcription. pM49, one of the vTFs that has not been studied before, was identified to be a component of the complex that interacts with pM95. In this study, we began to investigate the potential role of pM49 in viral late gene expression. A recombinant MCMV expressing C-terminal FLAG-tagged pM49 was constructed to study the expression kinetics and localization of pM49. pM49 was expressed at the late time of virus infection. Inhibition of viral DNA synthesis by phosphonate sodium phosphonic acid (PAA) abolished pM49 expression, indicating that it is a late protein. pM49 colocalized with pM44 at the viral replication compartment, similarly to other viral vTFs that have been reported. Mutant virus lacking full-length pM49 expression failed to express viral late genes, leading to nonproductive infection. The expression of immediate early and early genes was not affected, and viral DNA synthesis was only minimally affected during pM49-deficient virus infection. All of these data support the role of pM49 in viral late gene expression. After a series of mutagenesis analyses, two key residues, K325 and C326, were identified as required for pM49-pM95 interaction. Cells expressing pM49 with either single mutation of these two residues failed to rescue the late gene expression and support the replication of pM49-deficient virus. Our results indicated that pM49-pM95 interaction is essential for viral late gene expression.IMPORTANCE Cytomegalovirus (CMV) infections result in morbidity and mortality in immunocompromised individuals, and the virus is also a major cause of birth defects in newborns. Currently, because of the unavailability of vaccines against this virus and restricted antiviral therapies with low toxicity, as well as the emergency of resistant strain of this virus, the understanding of viral late gene regulation may provide clues to study new antiviral drugs or vaccines. In this study, we report that MCMV protein pM49 is critical for viral late gene transcription, based on its interaction with pM95. This finding reveals the important role of pM49-pM95 interaction in the regulation of viral late gene expression and that it could be a future potential target for therapeutic intervention in CMV diseases.

Facile and Modular Pipeline for Protein-Specific Antibody Customization.

Antibodies are fundamental tools for basic science; however, high-quality antibodies suitable for multiple experimental applications are often inaccessible to research laboratories. To this end, a modular and low-cost pipeline for small-scale antibody customization is developed. First, soluble antigens are designed according to the secondary structure of a desired protein. Then, the antigens are efficiently displayed on a modular nanoplatform by intein-mediated trans-splicing (TS) that enables elicitation of high titers of protein-specific antibodies. After that, target antibodies are obtained by a modular HaloLink resin platform with antigens as the ligand that is devised by intein-mediated TS. Finally, purified antibodies show excellent properties in immunofluorescence, immunoprecipitation, and western blotting assays. Overall, these results suggest that the proposed pipeline is amenable to the generation of high-quality, research-grade antibodies and to aid in protein functional studies.

Dissecting the Role of DDX21 in Regulating Human Cytomegalovirus Replication.

DDX21 regulates the biogenesis of rRNA and transcription of ribonucleoprotein genes. Recently, it has been reported that DDX21 regulates the growth of some RNA viruses through various mechanisms, such as inhibiting viral genome replication, suppressing virion assembly and release, and modulating antiviral immune responses (Chen et al., Cell Host Microbe 15:484-493, 2014, https://doi.org/10.1016/j.chom.2014.03.002; Dong et al., Biophys Res Commun, 473:648-653, 2016, https://doi.org/10.1016/j.bbrc.2016.03.120; and Watanabe et al., PLoS Pathog 5:e1000654, 2009, https://doi.org/10.1371/journal.ppat.1000654). The relationship between DDX21 and DNA viruses has not yet been explored. In this study, we used human cytomegalovirus (HCMV), a large human DNA virus, to investigate the potential role of DDX21 in DNA virus replication. We found that HCMV infection prevented the repression of DDX21 at protein and mRNA levels. Knockdown of DDX21 inhibited HCMV growth in human fibroblast cells (MRC5). Immunofluorescence and quantitative PCR (qPCR) results showed that knockdown of DDX21 did not affect viral DNA replication or the formation of the viral replication compartment but did significantly inhibit viral late gene transcription. Some studies have reported that DDX21 knockdown promotes the accumulation of R-loops that could restrain RNA polymerase II elongation and inhibit the transcription of certain genes. Thus, we used the DNA-RNA hybrid-specific S9.6 antibody to stain R-loops and observed that more R-loops formed in DDX21-knockdown cells than in control cells. Moreover, an DNA-RNA immunoprecipitation assay showed that more R-loops accumulated on a viral late gene in DDX21-knockdown cells. Altogether, these results suggest that DDX21 knockdown promotes the accumulation of R-loops, which prevents viral late gene transcription and consequently results in the suppression of HCMV growth. This finding provides new insight into the relationship between DDX21 and DNA virus replication.IMPORTANCE Previous studies have confirmed that DDX21 is vital for the regulation of various aspects of RNA virus replication. Our research is the first report on the role of DDX21 in HCMV DNA virus replication. We identified that DDX21 knockdown affected HCMV growth and viral late gene transcription. In order to elucidate how DDX21 regulated this transcription, we applied DNA-RNA immunoprecipitation by using the DNA-RNA hybrid-specific S9.6 antibody to test whether more R-loops accumulated on the viral late gene. Consistent with our expectation, more R-loops were detected on the viral late gene at late HCMV infection time points, which demonstrated that the accumulation of R-loops caused by DDX21 knockdown prevented viral late gene transcription and consequently impaired HCMV replication. These results reveal that DDX21 plays an important role in regulating HCMV replication and also provide a basis for investigating the role of DDX21 in regulating other DNA viruses.

Versatile Functionalization of Ferritin Nanoparticles by Intein-Mediated Trans-Splicing for Antigen/Adjuvant Co-delivery.

Self-assembling protein nanoparticles are extensively and increasingly engineered to integrate adjuvants with antigens to elicit potent and long-term immunity due to uniform architecture, inherent biocompatibility, and excellent plasticity. However, functionalization of nanoparticles by surface tailoring has two common problems: (1) disassembly caused by loaded cargoes; and (2) an adjuvant that is inconvenient to co-deliver with an antigen by genetic fusion. Here, we report an intein-mediated trans-splicing approach that overcomes the detrimental effects of loaded proteins on ferritin nanoparticle stability and allows concurrent display of antigen and adjuvant in a facile, efficient, and site-specific manner. An immunization study with an epitope-based model antigen reveals that antigen and adjuvant co-delivery nanoparticles induce a more potent protective immunity than other formulations do. Our results demonstrate that protein engineering represents an intriguing approach for antigen/adjuvant co-delivery to potentiate antigen-associated immune responses.

Murine Cytomegalovirus Protein pM91 Interacts with pM79 and Is Critical for Viral Late Gene Expression.

Viral gene expression is tightly regulated during cytomegalovirus (CMV) lytic replication, but the detailed mechanism of late gene transcription remains to be fully understood. Previous studies reported that six viral proteins (named viral transactivation factors [vTFs]) supporting late gene expression were conserved in beta- and gammaherpesviruses but not in alphaherpesviruses. Here, we performed coimmunoprecipitation experiments to elucidate the organization of these six proteins in murine CMV. Our results showed that these proteins formed a complex by both direct and indirect interactions. Specifically, pM91 strongly bound to pM79 even in the absence of other vTFs. Similar to pM79, pM91 exhibited early-late expression kinetics and localized within nuclear viral replication compartments during infection. Functional analysis was also performed using the pM91-deficient virus. Real-time PCR results revealed that abrogation of M91 expression markedly reduced viral late gene expression and progeny virus production without affecting viral DNA synthesis. Using mutagenesis, we found that residues E61, D62, D89, and D96 in pM91 were required for the pM91-pM79 interaction. Disruption of the interaction via E61A/D62A or D89A/D96A double mutation in the context of virus infection inhibited progeny virus production. Our data indicate that pM91 is a component of the viral late gene transcription factor complex and that the pM91-pM79 interaction is essential for viral late gene expression.IMPORTANCE Cytomegalovirus (CMV) infection is the leading cause of birth defects and causes morbidity and mortality in immunocompromised patients. The regulation of viral late gene transcription is not well elucidated, and understanding of this process benefits the development of novel therapeutics against CMV infection. This study (i) identified that six viral transactivation factors encoded by murine CMV form a complex, (ii) demonstrated that pM91 interacts with pM79 and that pM91 and pM79 colocalize in the nuclear viral replication compartments, (iii) confirmed that pM91 is critical for viral late gene expression but dispensable for viral DNA replication, and (iv) revealed that the pM91-pM79 interaction is required for progeny virus production. These findings give an explanation of how CMV regulates late gene expression and have important implications for the design of antiviral strategies.

A Modular Vaccine Development Platform Based on Sortase-Mediated Site-Specific Tagging of Antigens onto Virus-Like Particles.

Virus-like particles (VLPs) can be used as powerful nanoscale weapons to fight against virus infection. In addition to direct use as vaccines, VLPs have been extensively exploited as platforms on which to display foreign antigens for prophylactic vaccination and immunotherapeutic treatment. Unfortunately, fabrication of new chimeric VLP vaccines in a versatile, site-specific and highly efficient manner is beyond the capability of traditional VLP vaccine design approaches, genetic insertion and chemical conjugation. In this study, we described a greatly improved VLP display strategy by chemoenzymatic site-specific tailoring antigens on VLPs surface with high efficiency. Through the transpeptidation mediated by sortase A, one protein and two epitopes containing N-terminal oligoglycine were conjugated to the LPET motif on the surface of hepatitis B virus core protein (HBc) VLPs with high density. All of the new chimeric VLPs induced strong specific IgG responses. Furthermore, the chimeric VLPs with sortase A tagged enterovirus 71 (EV71) SP70 epitope could elicit effective antibodies against EV71 lethal challenging as well as the genetic insertion chimeric VLPs. The sortase A mediated chemoenzymatic site-specific tailoring of the HBc VLP approach shows great potential in new VLP vaccine design for its simplicity, site specificity, high efficiency, and versatility.