A recombinant virus-like particle vaccine against adenovirus-7 induces a potent humoral response.

Adenoviruses (AdVs) cause infections in humans that range from mild to severe, and can cause outbreaks particularly in close contact settings. Several human AdV types have been identified, which can cause a wide array of clinical manifestations. AdV types 4 and 7 (AdV-4 and AdV-7), which are among the most commonly circulating types in the United States, are known to cause acute respiratory disease that can result in hospitalization and rarely, death. Currently, the only vaccines approved for use in humans are live virus vaccines against AdV-4 and AdV-7, though these vaccines are only authorized for use in U.S. military personnel. While they are efficacious, use of these live virus vaccines carries considerable risks of vaccine-associated viral shedding and recombination. Here, we present an alternative vaccination strategy against AdV-7 using the virus-like particle platform (AdVLP-7). We describe the production of stable recombinant AdVLP-7, and demonstrate that AdVLP-7 is structurally analogous to wild-type AdV-7 virions (WT AdV-7). Preclinical immunogenicity studies in mice show that AdVLP-7 elicits a potent humoral immune response, comparable to that observed in mice immunized with WT AdV-7. Specifically, AdVLP-7 induces high titers of antibodies against AdV-7-specific antigens that can effectively neutralize AdV-7.

Novel and efficient method for the reconstruction of adenoviruses through isothermal assembly and its potential applications.

Adenovirus based vectors are useful tools for vaccine development, gene therapy, and oncolytic virotherapy. Here we describe a novel approach for the genetic engineering of any portion of the adenovirus genome and the reconstruction of either fully replication competent or defective virions. This innovative strategy is rapid, effective and suitable for the manipulation of the entire genome broadening the spectrum of potential applications for the adenovirus system. Our strategy involved insertion of restriction enzyme recognition sequences absent in the native virus into the termini of the adenovirus genome in order to facilitate recovery. These restriction enzyme sites, together with the two inverted terminal repeats and packaging sequences, were synthesized and then subcloned into the pBR322 vector. The remaining internal portion of the adenovirus genome was separated and amplified via PCR into six segments, of which groups of two were joined together by PCR and then subcloned into pBR322 plasmids. During the PCR reaction, an overlap of 30-40 bp was added to the termini of the adjacent fragments, allowing for the subsequent isothermal assembly and correct orientation of all fragments. This approach allows for the genetic modification of each genomic fragment according to the specific research goals, (e.g., deletion, substitution, addition, etc.) To recreate the entire viral genome, the four engineered fragments (each comprised of two adenovirus genomic sections) as well as the pBR322 backbone, were reassembled into a single construct utilizing an isothermal assembly reaction. Finally, the reassembled plasmid containing the entire genome was linearized and transfected into HEK293 cells to recover the complete reconstructed adenoviral vector. Using this approach, we have successfully generated two recombinant reporter adenoviruses, one of human adenovirus serotype 14 and another of serotype 55. The E3 region was replaced by the reporter genes (GFP and Luciferase) to visualize and track the recovery process. Subsequent infection of A549 cells with these reconstructed adenovirus vectors demonstrated that they were replication competent. This method shortens the viral reconstruction process because the one-step isothermal assembly requires less than 4 days, and recombinant adenovirus recovery occurs within 10 days. This new method allows for single or multiple genetic modifications within any portion of the viral genome and can be used to construct or manipulate any adenovirus whose complete genome sequence is known.

Immunogenicity and Efficacy of Monovalent and Bivalent Formulations of a Virus-Like Particle Vaccine against SARS-CoV-2.

Virus-like particles (VLPs) offer great potential as a safe and effective vaccine platform against SARS-CoV-2, the causative agent of COVID-19. Here, we show that SARS-CoV-2 VLPs can be generated by expression of the four viral structural proteins in a mammalian expression system. Immunization of mice with a monovalent VLP vaccine elicited a potent humoral response, showing neutralizing activity against multiple variants of SARS-CoV-2. Subsequent immunogenicity and efficacy studies were performed in the Golden Syrian hamster model, which closely resembles the pathology and progression of COVID-19 in humans. Hamsters immunized with a bivalent VLP vaccine were significantly protected from infection with the Beta or Delta variant of SARS-CoV-2. Vaccinated hamsters showed reduced viral load, shedding, replication, and pathology in the respiratory tract. Immunized hamsters also showed variable levels of cross-neutralizing activity against the Omicron variant. Overall, the VLP vaccine elicited robust protective efficacy against SARS-CoV-2. These promising results warrant further study of multivalent VLP vaccines in Phase I clinical trials in humans.

The Alginate and Motility Regulator AmrZ is Essential for the Regulation of the Dispersion Response by Pseudomonas aeruginosa Biofilms.

Dispersion is an active process exhibited by Pseudomonas aeruginosa during the late stages of biofilm development or in response to various cues, including nitric oxide and glutamate. Upon cue sensing, biofilm cells employ enzymes that actively degrade the extracellular matrix, thereby allowing individual cells to become liberated. While the mechanism by which P. aeruginosa senses and relays dispersion cues has been characterized, little is known about how dispersion cue sensing mechanisms result in matrix degradation. Considering that the alginate and motility regulator AmrZ has been reported to regulate genes that play a role in dispersion, including those affecting virulence, c-di-GMP levels, Pel and Psl abundance, and motility, we asked whether AmrZ contributes to the regulation of dispersion. amrZ was found to be significantly increased in transcript abundance under dispersion-inducing conditions, with the inactivation of amrZ impairing dispersion by P. aeruginosa biofilms in response to glutamate and nitric oxide. While the overexpression of genes encoding matrix-degrading enzymes pelA, pslG, and/or endA resulted in the dispersion of wild-type biofilms, similar conditions failed to disperse biofilms formed by dtamrZ. Likewise, the inactivation of amrZ abrogated the hyperdispersive phenotype of PAO1/pJN-bdlA_G31A biofilms, with dtamrZ-impaired dispersion being independent of the expression, production, and activation of BdlA. Instead, dispersion was found to require the AmrZ-target genes napB and PA1891. Our findings indicate that AmrZ is essential for the regulation of dispersion by P. aeruginosa biofilms, functions downstream of BdlA postdispersion cue sensing, and regulates the expression of genes contributing to biofilm matrix degradation as well as napB and PA1891. IMPORTANCE In P. aeruginosa, biofilm dispersion has been well-characterized with respect to dispersion cue perception, matrix degradation, and the consequences of dispersion. While the intracellular signaling molecule c-di-GMP has been linked to many of the phenotypic changes ascribed to dispersion, including the modulation of motility and matrix production, little is known about the regulatory mechanisms leading to matrix degradation and cells actively leaving the biofilm. In this study, we report for the first time an essential role of the transcriptional regulator AmrZ and two AmrZ-dependent genes, napB, and PA1891, in the dispersion response, thereby linking dispersion cue sensing via BdlA to the regulation of matrix degradation and to the ultimate liberation of bacterial cells from the biofilm.