Human Adenovirus Type 26 Infection Mediated by αvß3 Integrin Is Caveolin-1-Dependent.

Human adenovirus type 26 (HAdV26) has been recognized as a promising platform for vaccine vector development, and very recently vaccine against COVID-19 based on HAdV26 was authorized for emergency use. Nevertheless, basic biology of this virus, namely, pathway which HAdV26 uses to enter the cell, is still insufficiently known. We have shown here that HAdV26 infection of human epithelial cells expressing low amount of αvß3 integrin involves clathrin and is caveolin-1-independent, while HAdV26 infection of cells with high amount of αvß3 integrin does not involve clathrin but is caveolin-1-dependent. Thus, this study demonstrates that caveolin-1 is limiting factor in αvß3 integrin-mediated HAdV26 infection. Regardless of αvß3 integrin expression, HAdV26 infection involves dynamin-2. Our data provide for the first-time description of HAdV26 cell entry pathway, hence increase our knowledge of HAdV26 infection. Knowing that functionality of adenovirus vector is influenced by its cell entry pathway and intracellular trafficking, our results will contribute to better understanding of HAdV26 immunogenicity and antigen presentation when used as vaccine vector. IMPORTANCE In order to fulfill its role as a vector, adenovirus needs to successfully deliver its DNA genome to the host nucleus, a process highly influenced by adenovirus intracellular translocation. Thus, cell entry pathway and intracellular trafficking determine functionality of human adenovirus-based vectors. Endocytosis of HAdV26, currently extensively studied as a vaccine vector, has not been described so far. We present here that HAdV26 infection of human epithelial cells with high expression of αvß3 integrin, one of the putative HAdV26 receptors, is caveolin-1- and partially dynamin-2-dependent. Since caveolin containing domains provide a unique environment for specific signaling events and participate in inflammatory signaling one can imagine that directing HAdV26 cell entry toward caveolin-1-mediate pathway might play role in immunogenicity of this virus. Therefore, our results contribute to better understanding of HAdV26 infection pathway, hence, can be helpful in explaining induction of immune response and antigen presentation by HAdV26-based vaccine vector.

A Human In Vitro Model to Study Adenoviral Receptors and Virus Cell Interactions.

To develop adenoviral cell- or tissue-specific gene delivery, understanding of the infection mechanisms of adenoviruses is crucial. Several adenoviral attachment proteins such as CD46, CAR and sialic acid have been identified and studied. However, most receptor studies were performed on non-human cells. Combining our reporter gene-tagged adenovirus library with an in vitro human gene knockout model, we performed a systematic analysis of receptor usage comparing different adenoviruses side-by-side. The CRISPR/Cas9 system was used to knockout CD46 and CAR in the human lung epithelial carcinoma cell line A549. Knockout cells were infected with 22 luciferase-expressing adenoviruses derived from adenovirus species B, C, D and E. HAdV-B16, -B21 and -B50 from species B1 as well as HAdV-B34 and -B35 were found to be CD46-dependent. HAdV-C5 and HAdV-E4 from species E were found to be CAR-dependent. Regarding cell entry of HAdV-B3 and -B14 and all species D viruses, both CAR and CD46 play a role, and here, other receptors or attachment structures may also be important since transductions were reduced but not completely inhibited. The established human knockout cell model enables the identification of the most applicable adenovirus types for gene therapy and to further understand adenovirus infection biology.

Dual-Antigen COVID-19 Vaccine Subcutaneous Prime Delivery With Oral Boosts Protects NHP Against SARS-CoV-2 Challenge.

We have developed a dual-antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike protein (S-Fusion) and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to increase the potential for MHC class II responses. The vaccine antigens are delivered by a human adenovirus serotype 5 platform, hAd5 [E1-, E2b-, E3-], previously demonstrated to be effective in the presence of Ad immunity. Vaccination of rhesus macaques with the hAd5 S-Fusion + N-ETSD vaccine by subcutaneous prime injection followed by two oral boosts elicited neutralizing anti-S IgG and T helper cell 1-biased T-cell responses to both S and N that protected the upper and lower respiratory tracts from high titer (1 x 106 TCID50) SARS-CoV-2 challenge. Notably, viral replication was inhibited within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge.

Precursors of Viral Proteases as Distinct Drug Targets.

Viral proteases are indispensable for successful virion maturation, thus making them a prominent drug target. Their enzyme activity is tightly spatiotemporally regulated by expression in the precursor form with little or no activity, followed by activation via autoprocessing. These cleavage events are frequently triggered upon transportation to a specific compartment inside the host cell. Typically, precursor oligomerization or the presence of a co-factor is needed for activation. A detailed understanding of these mechanisms will allow ligands with non-canonical mechanisms of action to be designed, which would specifically modulate the initial irreversible steps of viral protease autoactivation. Binding sites exclusive to the precursor, including binding sites beyond the protease domain, can be exploited. Both inhibition and up-regulation of the proteolytic activity of viral proteases can be detrimental for the virus. All these possibilities are discussed using examples of medically relevant viruses including herpesviruses, adenoviruses, retroviruses, picornaviruses, caliciviruses, togaviruses, flaviviruses, and coronaviruses.

Human species D adenovirus hexon capsid protein mediates cell entry through a direct interaction with CD46.

Human adenovirus species D (HAdV-D) types are currently being explored as vaccine vectors for coronavirus disease 2019 (COVID-19) and other severe infectious diseases. The efficacy of such vector-based vaccines depends on functional interactions with receptors on host cells. Adenoviruses of different species are assumed to enter host cells mainly by interactions between the knob domain of the protruding fiber capsid protein and cellular receptors. Using a cell-based receptor-screening assay, we identified CD46 as a receptor for HAdV-D56. The function of CD46 was validated in infection experiments using cells lacking and overexpressing CD46, and by competition infection experiments using soluble CD46. Remarkably, unlike HAdV-B types that engage CD46 through interactions with the knob domain of the fiber protein, HAdV-D types infect host cells through a direct interaction between CD46 and the hexon protein. Soluble hexon proteins (but not fiber knob) inhibited HAdV-D56 infection, and surface plasmon analyses demonstrated that CD46 binds to HAdV-D hexon (but not fiber knob) proteins. Cryoelectron microscopy analysis of the HAdV-D56 virion-CD46 complex confirmed the interaction and showed that CD46 binds to the central cavity of hexon trimers. Finally, soluble CD46 inhibited infection by 16 out of 17 investigated HAdV-D types, suggesting that CD46 is an important receptor for a large group of adenoviruses. In conclusion, this study identifies a noncanonical entry mechanism used by human adenoviruses, which adds to the knowledge of adenovirus biology and can also be useful for development of adenovirus-based vaccine vectors.