Directed evolution of human Adenoviruses to improve vectored vaccines properties

Replication incompetent human Adeno (hAd)-based vectors are able to induce potent adaptive immune responses in animal models and humans, being of interest for both infectious diseases and cancer therapy or prevention. Different Adenovirus-based vaccines have been approved for human use, including those developed to contrast Covid-19 pandemic. Immunological potency of hAdvectors based on different serotypes can be influenced by the extent of pre-existing host immunity, different cell tropism and intracellular trafficking, also impacting the extent of innate immunity induction. Vaccines based on species C hAd are among the most potent, yet significant seroprevalence in humans limits their application into the clinic, with pre-existing neutralizing antibodies negatively affecting vectored vaccine immunogenicity and efficacy. Error prone PCR represents an efficient method to introduce random mutations by reducing the DNA polymerase fidelity, allowing directed evolution and improvement of target proteins. Since the majority of hAd neutralization determinants are located in the hypervariable regions (HVR) of the hexon, the major capsid protein, we generated seven different hexon-variant libraries with mutations in single or multiple HVRs from Group C hAd, through error prone PCR. Such libraries have been used to produce complex repertoires of hAd vectors potentially including variants with novel properties such as lower recognition by anti-hAd antibodies present in humans or reduced unwanted side effects in the vaccinees. These libraries will be surveyed with different screening strategies to identify novel variants with improved properties for both prophylactic and therapeutic vaccine applications.

SARS CoV-2;a closer look at the agent of the pandemic of modern times

Due to the COVID-19 pandemic, as of September 2021, a total of 222,309,456 people were infected in the world and a total of 4,592,685 patients were lost. The pandemic, which has a fatality rate of around 2%, has made and continues to make us live thhrough all experiences of epidemics that we have only read about in Annals of Medicine and Microbiology and that deeply affected the World at their times. The virus causing the pandemic has a positive polarity RNA genome of 30,000 bases and produces a total of 29 proteins. Of these proteins, 4 are structural, 16 are nonstructural, and 9 are accessory proteins. SARS-CoV-2 is an enveloped RNA virus with a diameter of 150-200 nm, has an S (spike-spike-tassel) glycoprotein on its surface, which, like other coronaviruses, creates the crown appearance unique to these viruses. After the S protein is synthesized as a polyprotein, it is cleaved into S1 and S2 subunits. The S1 subunit binds to the target cell, and the S2 subunit performs fusion with the cell membrane to be infected. Since these functions are critical features of a successful viral infection, the S protein is the main target of all interventions to prevent virus infection. In this context, the main target of neutralizing antibodies and drugs to stop virus infection before it starts is the S protein. The S protein has a trimer structure similar to hemagglutinin in influenza virus and contains the fusion peptide that becomes exposed during transition from the prefusion configuration to the fusion configuration and facilitates the fusion function with the cellular/endosomal membranes. Apart from the S protein, SARS-CoV-2 has structural proteins known as E (envelope), M (membrane), and N (nucleocapsid) proteins;The N protein binds to the RNA genome and together with the S, E and M proteins and the RNA genome form the virion. While SARS-CoV-2 S protein attaches to cells using Cellular Angiotensin Converting Enzyme 2 (HCoV- NL63, SARS-CoV and SARS-CoV-2), other coronaviruses use different receptors (Aminopeptidase N-HCoV-229E;dipeptidyl peptidase 4- MERS-CoV). Unlike viruses in this group, the SARS CoV-2 S1 protein with receptor binding domain (RBD) has a cleavage site made up of polybasic amino acids at the S1-S2 border and used by the cellular furin protease, which is believed to provide advantages to the virus in proteolytic cleavage, cell tropism, virulence and pathogenicity. ACE-2 is important in the renin-angiotensin-aldosterone system and although it is rarely found in the circulation, it is widely expressed in organs and is an enzyme involved in the regulation of blood pressure and fluid balance. Following intracellular entry and fusion of membranes, the SARS-CoV-2 genome is released into the cytoplasm and gene expression proceeds as a temporally and spatially well-regulated process. Non-structural proteins, which are produced from direct translation of ORF1a and ORF1b regions of positive sense genomic RNA, form the replication and transcription complex. These complexes establish the infrastructure for the next steps. The common features of coronaviruses such as cytoplasmic replication, viral gene expression through sub-genomic nested set messages, exocytosis of mature virions within vesicles occur in SARS-CoV-2 as well. One of the most important problems in the COVID-19 pandemic has been the emergence of variant viruses. These viruses adversely affecting the transmission rate, virulence, clinical course, and the effectiveness of the diagnostic or therapeutic methods carry mutations that lead to amino acid changes, especially in the RBD region. The World Health Organization and other authorities refer to these viruses as variants of concern or variants of interest. As of September 2021, WHO lists Alpha (UK, September 2020), Beta (South Africa, May 2020), Gamma (Brazil, November 2020), and Delta (India, October 2020) viruses as variants of concern. Also, Eta (December 2020), Iota (USA, November 2020), Kappa (India, October 2020), Lambda (Peru December, 2020) and Mu (Colombia, January 2021) mutant viruses are on he list variants of interest. In conclusion, less than 2 years of time has passed since the emergence of the COVID-19 agent SARS CoV-2 virus. However, this virus has been the most extensively studied viral agent in the history of medicine and the most detailed information has been gathered about the infection. Despite all these, it is difficult to indicate that the fight against this pathogen has been successful nor are we any closer to declare that the enormous danger the virus poses to humanity is reduced.

Construction, Characterization and Application of Recombinant Porcine Deltacoronavirus Expressing Nanoluciferase.

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhoea in suckling piglets and has the potential for cross-species transmission. No effective PDCoV vaccines or antiviral drugs are currently available. Here, we successfully generated an infectious clone of PDCoV strain CHN-HN-2014 using a combination of bacterial artificial chromosome (BAC)-based reverse genetics system with a one-step homologous recombination. The recued virus (rCHN-HN-2014) possesses similar growth characteristics to the parental virus in vitro. Based on the established infectious clone and CRISPR/Cas9 technology, a PDCoV reporter virus expressing nanoluciferase (Nluc) was constructed by replacing the NS6 gene. Using two drugs, lycorine and resveratrol, we found that the Nluc reporter virus exhibited high sensibility and easy quantification to rapid antiviral screening. We further used the Nluc reporter virus to test the susceptibility of different cell lines to PDCoV and found that cell lines derived from various host species, including human, swine, cattle and monkey enables PDCoV replication, broadening our understanding of the PDCoV cell tropism range. Taken together, our reporter viruses are available to high throughput screening for antiviral drugs and uncover the infectivity of PDCoV in various cells, which will accelerate our understanding of PDCoV.

Broad Severe Acute Respiratory Syndrome Coronavirus 2 Cell Tropism and Immunopathology in Lung Tissues From Fatal Coronavirus Disease 2019.

BACKGROUND: Coronavirus disease 2019 (COVID-19) patients manifest with pulmonary symptoms reflected by diffuse alveolar damage (DAD), excessive inflammation, and thromboembolism. The mechanisms mediating these processes remain unclear. METHODS: We performed multicolor staining for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins and lineage markers to define viral tropism and lung pathobiology in 5 autopsy cases. RESULTS: Lung parenchyma showed severe DAD with thromboemboli. Viral infection was found in an extensive range of cells including pneumocyte type II, ciliated, goblet, club-like, and endothelial cells. More than 90% of infiltrating immune cells were positive for viral proteins including macrophages, monocytes, neutrophils, natural killer (NK) cells, B cells, and T cells. Most but not all infected cells were angiotensin-converting enzyme 2 (ACE2) positive. The numbers of infected and ACE2-positive cells are associated with extensive tissue damage. Infected tissues exhibited high levels of inflammatory cells including macrophages, monocytes, neutrophils, and NK cells, and low levels of B cells but abundant T cells consisting of mainly T helper cells, few cytotoxic T cells, and no regulatory T cells. Robust interleukin-6 expression was present in most cells, with or without infection. CONCLUSIONS: In fatal COVID-19 lungs, there are broad SARS-CoV-2 cell tropisms, extensive infiltrated innate immune cells, and activation and depletion of adaptive immune cells, contributing to severe tissue damage, thromboemboli, excess inflammation, and compromised immune responses.

Host metabolism dysregulation and cell tropism identification in human airway and alveolar organoids upon SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is spread primary via respiratory droplets and infects the lungs. Currently widely used cell lines and animals are unable to accurately mimic human physiological conditions because of the abnormal status of cell lines (transformed or cancer cells) and species differences between animals and humans. Organoids are stem cell-derived self-organized three-dimensional culture in vitro and model the physiological conditions of natural organs. Here we showed that SARS-CoV-2 infected and extensively replicated in human embryonic stem cells (hESCs)-derived lung organoids, including airway and alveolar organoids which covered the complete infection and spread route for SARS-CoV-2 within lungs. The infected cells were ciliated, club, and alveolar type 2 (AT2) cells, which were sequentially located from the proximal to the distal airway and terminal alveoli, respectively. Additionally, RNA-seq revealed early cell response to virus infection including an unexpected downregulation of the metabolic processes, especially lipid metabolism, in addition to the well-known upregulation of immune response. Further, Remdesivir and a human neutralizing antibody potently inhibited SARS-CoV-2 replication in lung organoids. Therefore, human lung organoids can serve as a pathophysiological model to investigate the underlying mechanism of SARS-CoV-2 infection and to discover and test therapeutic drugs for COVID-19.

Priming of SARS-CoV-2 S protein by several membrane-bound serine proteinases could explain enhanced viral infectivity and systemic COVID-19 infection.

The ongoing COVID-19 pandemic has already caused over a million deaths worldwide, and this death toll will be much higher before effective treatments and vaccines are available. The causative agent of the disease, the coronavirus SARS-CoV-2, shows important similarities with the previously emerged SARS-CoV-1, but also striking differences. First, SARS-CoV-2 possesses a significantly higher transmission rate and infectivity than SARS-CoV-1 and has infected in a few months over 60 million people. Moreover, COVID-19 has a systemic character, as in addition to the lungs, it also affects the heart, liver, and kidneys among other organs of the patients and causes frequent thrombotic and neurological complications. In fact, the term "viral sepsis" has been recently coined to describe the clinical observations. Here I review current structure-function information on the viral spike proteins and the membrane fusion process to provide plausible explanations for these observations. I hypothesize that several membrane-associated serine proteinases (MASPs), in synergy with or in place of TMPRSS2, contribute to activate the SARS-CoV-2 spike protein. Relative concentrations of the attachment receptor, ACE2, MASPs, their endogenous inhibitors (the Kunitz-type transmembrane inhibitors, HAI-1/SPINT1 and HAI-2/SPINT2, as well as major circulating serpins) would determine the infection rate of host cells. The exclusive or predominant expression of major MASPs in specific human organs suggests a direct role of these proteinases in e.g., heart infection and myocardial injury, liver dysfunction, kidney damage, as well as neurological complications. Thorough consideration of these factors could have a positive impact on the control of the current COVID-19 pandemic.