Covalently closed circular DNA (cccDNA) exists as a stable episomal minichromosome in the nucleus of hepatocytes and is responsible for hepatitis B virus (HBV) persistence. We recently reported a technique involving recombinant cccDNA (rcccDNA) of HBV by site-specific DNA recombination. A floxed monomeric HBV genome was engineered into a precursor plasmid (prcccDNA) which was excised via Cre/loxP-mediated DNA recombination to form a 3.3-kb rcccDNA bearing a loxP-chimeric intron. The foreign sequence was efficiently removed during RNA splicing, rendering a functionally seamless insertion. We characterized rcccDNA formation, effective viral transcription, and replication induced by rcccDNA both in vitro and in vivo. Furthermore, we closely simulated chronic hepatitis by using a replication-defective recombinant adenoviral vector to deliver rcccDNA to the transgenic mice expressing Cre recombinase, which led to prominent HBV persistence. Here, we describe a detailed protocol about how to construct and evaluate Cre/loxP-based recombinant HBV cccDNA system both in vitro and in vivo.

The variable domain of a heavy-chain antibody (VHH) has the potential to be used to redirect the cell tropism of adenoviral vectors. Here, we attempted to establish platforms to simplify the screening of VHHs for their specific targeting function when being incorporated into the fiber of adenovirus. Both fowl adenovirus 4 (FAdV-4) and simian adenovirus 1 (SAdV-1) have two types of fiber, one of which is dispensable for virus propagation and is a proper site for VHH display. An intermediate plasmid, pMD-FAV4Fs, was constructed as the start plasmid for FAdV-4 fiber2 modification. Foldon from phage T4 fibritin, a trigger for trimerization, was employed to bridge the tail/shaft domain of fiber2 and VHHs against human CD16A, a key membrane marker of natural killer (NK) cells. Through one step of restriction-assembly, the modified fiber2 was transferred to the adenoviral plasmid, which was linearized and transfected to packaging cells. Five FAdV-4 viruses carrying the GFP gene were finally rescued and amplified, with three VHHs being displayed. One recombinant virus, FAdV4FC21-EG, could hardly transduce human 293 or Jurkat cells. In contrast, when it was used at a multiplicity of infection of 1000 viral particles per cell, the transduction efficiency reached 51% or 34% for 293 or Jurkat cells expressing exogenous CD16A. Such a strategy of fiber modification was transplanted to the SAdV-1 vector to construct SAdV1FC28H-EG, which moderately transduced primary human NK cells while the parental virus transduced none. Collectively, we reformed the strategy of integrating VHH to fiber and established novel platforms for screening VHHs to construct adenoviral vectors with a specific tropism.

OBJECTIVE: Porcine epidemic diarrhea (PED) is a highly infectious and economically significant gastrointestinal disorder that affects pigs of all ages. Preventing and controlling PED is achieved by immunizing sows with vaccines, enabling passive piglet immunization via colostrum. The prevalence of G2b porcine epidemic diarrhea virus (PEDV) continues in China despite the use of commercial vaccines, raising questions regarding current vaccine efficacy and the need for novel vaccine development. Adenovirus serotype 5 (Ad5) has several advantages, including high transduction efficiency, a wide range of host cells, and the ability to infect cells at various stages. In this study, we expressed the immunogenic proteins of spike (S) using an Ad5 vector and generated a PED vaccine candidate by inducing significant humoral immunity. The rAd5-PEDV-S prevented PED-induced weight loss, diarrhea, and intestinal damage in piglets. This novel vaccine candidate strain possesses the potential for use in the pig breeding industry.

Classic chimeric hemagglutinin (cHA) was designed to induce immune responses against the conserved stalk domain of HA. However, it is unclear whether combining more than one HA head domain onto one stalk domain is immunogenic and further induce immune responses against influenza viruses. Here, we constructed numerous novel cHAs comprising two or three fuzed head domains from different subtypes grafted onto one stalk domain, designated as cH1-H3, cH1-H7, cH1-H3-H7, and cH1-H7-H3. The three-dimensional structures of these novel cHAs were modelled using bioinformatics simulations. Structural analysis showed that the intact neutralizing epitopes were exposed in cH1-H7 and were predicted to be immunogenic. The immunogenicity of the cHAs constructs was evaluated in mice using a chimpanzee adenoviral vector (AdC68) vaccine platform. The results demonstrated that cH1-H7 expressed by AdC68 (AdC68-cH1-H7) induced the production of high levels of binding antibodies, neutralizing antibodies, and hemagglutinin inhibition antibodies against homologous pandemic H1N1, drifted seasonal H1N1, and H7N9 virus. Moreover, vaccinated mice were fully protected from a lethal challenge with the aforementioned influenza viruses. Hence, cH1-H7 cHAs with potent immunogenicity might be a potential novel vaccine to provide protection against different subtypes of influenza virus.

OBJECTIVE: The essential goal of vaccination is to generate potent and long-term protection against diseases. Several factors including vaccine vector, delivery route, and boosting regimen influence the outcome of prime-boost immunization approaches. The immunization regimens by constructing a novel simian adenovirus-vectored COVID-19 vaccine and employing combination of intranasal and intramuscular inoculations could elicit mucosal neutralizing antibodies against five mutant strains in the respiratory tract and strong systemic immunity. Immune protection could last for more than 32 weeks. Vectored vaccine construction and immunization regimens have positively impacted respiratory disease prevention.

The development of an efficient and safe coronavirus disease 2019 (COVID-19) vaccine is a crucial approach for managing the severe acute respiratory disease coronavirus 2 (SARS-CoV-2) pandemic in light of current conditions. In this study, we produced a shortened segment of the optimized SARS-CoV-2 spike gene (2043 bp, termed S1) that was able to encode a truncated S1 protein. The protein was tested to determine if it could elicit efficient immunization in mice against SARS-CoV-2. The presence of the S1 protein was confirmed with immunofluorescence and Western blotting. An adenovirus vaccine bearing the S1 gene fragment (Ad-S1) was administered intramuscularly to mice four times over 4 weeks. SARS-CoV-2 S1 protein humoral immunity was demonstrated in all immunized mice. The serum from immunized mice demonstrated excellent anti-infection activity in vitro. A robust humoral immune response against SARS-CoV-2 was observed in the mice after vaccination with Ad-S1, suggesting that the adenovirus vaccine may aid the development of vaccines against SARS-CoV-2 and other genetically distinct viruses.

Immune checkpoint blockade based on antibodies has shown great clinical success in patients, but the transitory working manner leads to restricted therapeutic benefits. Herein, a genetically engineered adenovirus is developed as the vector to deliver CRISPR/Cas9 (sgCas9-AdV) to achieve permanent PD-L1 gene editing with efficiency up to 78.7% exemplified in Hepa 1-6 liver cancer cells. Furthermore, the sgCas9-AdV is loaded into hydrogel made by silk fiber (SgCas9-AdV/Gel) for in vivo application. The silk-gel not only promotes local retention of sgCas9-AdV in tumor tissue, but also masks them from host immune system, thus ensuring effectively gene transduction over 9 days. Bearing these advantages, the sgCas9-AdV/Gel inhibits Hepa 1-6 tumor growth with 100% response rate by single-dose injection, through efficient PD-L1 disruption to elicit a T cell-mediated antitumor response. In addition, the sgCas9-AdV/Gel is also successfully extended into other refractory tumors. In CT26 colon tumor characterized by poor response to anti-PD-L1, sgCas9-AdV/Gel is demonstrated to competent and superior anti-PD-L1 antibody to suppress tumor progression. In highly aggressive orthotopic 4T1 mouse breast tumor, such a therapeutic paradigm significantly inhibits primary tumor growth and induces a durable immune response against tumor relapse/metastasis. Thus, this study provides an attractive and universal strategy for immunotherapy.

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The emerging SARS-CoV-2 variants of concern (VOCs) threaten the effectiveness of current COVID-19 vaccines administered intramuscularly and designed to only target the spike protein. There is a pressing need to develop next-generation vaccine strategies for broader and long-lasting protection. Using adenoviral vectors (Ad) of human and chimpanzee origin, we evaluated Ad-vectored trivalent COVID-19 vaccines expressing spike-1, nucleocapsid, and RdRp antigens in murine models. We show that single-dose intranasal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the tripartite protective immunity consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells and mucosal trained innate immunity. We further show that intranasal immunization provides protection against both the ancestral SARS-CoV-2 and two VOC, B.1.1.7 and B.1.351. Our findings indicate that respiratory mucosal delivery of Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy to induce all-around mucosal immunity against current and future VOC.

OBJECTIVE: Hypoxic-ischemic brain damage (HIBD) is a major cause of brain injury in neonates. Bone marrow mesenchymal stem cells (BMSCs) show therapeutic potential for HIBD, and genetic modification may enhance their neuroprotective effects. The goal of this study was to investigate the neuroprotective effects of hepatocyte growth factor (HGF)-overexpressing BMSCs (BMSCs-HGF) against HIBD and their underlying mechanisms.
METHODS: BMSCs were transfected with HGF using adenoviral vectors. HIBD models were established and then BMSCs were transplanted into the brains of HIBD rats via intraventricular injection. 2,3,5-Triphenyltetrazolium chloride (TTC) staining was used to measure cerebral infarction volumes. In vitro, primary cultured cortical neurons were co-cultured with BMSCs in a Transwell plate system. Oxygen-glucose deprivation (OGD) was applied to imitate hypoxic-ischemic insult, and PD98059 was added to the culture medium to block the phosphorylation of extracellular signal-regulated kinase (ERK). Cell apoptosis was determined using TUNEL staining. The expression of HGF was measured by immunofluorescence, real-time quantitative PCR (RT-qPCR), and western blots. The expression of phosphorylated ERK (p-ERK) and B-cell lymphoma-2 (Bcl-2) was measured by western blots.
RESULTS: HGF-gene transfection promoted BMSC proliferation. Moreover, BMSCs-HGF decreased HIBD-induced cerebral infarction volumes and enhanced the protective effects of the BMSCs against HIBD. BMSCs-HGF also increased expression of HGF, p-ERK, and Bcl-2 in brain tissues. In vitro, BMSC-HGF protected neurons against OGD-induced apoptosis. Inhibition of ERK phosphorylation abolished the neuroprotective effect of BMSCs-HGF against OGD.
CONCLUSIONS: BMSCs-HGF is a potential treatment for HIBD and that the ERK/Bcl-2 pathway is involved in the underlying neuroprotective mechanism.