Towards a Mathematical Model for the Viral Progression in the Pharynx

In this work, a comprehensive model for the viral progression in the pharynx has been developed. This one-dimension model considers both Fickian diffusion and convective flow coupled with chemical reactions, such as virus population growth, infected and uninfected cell accumulation as well as virus clearance. The effect of a sterilizing agent such as an alcoholic solution on the viral progression in the pharynx was taken into account and a parametric analysis for the effect of kinetic rate parameters on virus propagation was made. Moreover, different conditions caused by further medical treatment, such as a decrease in virus yield per infected cell, were examined. It is shown that the infection fails to establish by decreasing the virus yield per infected cell. It is believed that this work could be used to further investigate the medical treatment of viral progression in the pharynx.

Upstream process optimization and micro- and macrocarrier screening for large-scale production of the oncolytic H-1 protoparvovirus

The oncolytic virus H-1PV is a promising candidate for various cancer treatments. Therefore, production process needs to be optimized and scaled up for future market release. Currently, the virus is produced with minimum essential medium in 10-layer CellSTACK® chambers with limited scalability, requiring a minimum seeding density of 7.9E3 cells/cm2. Production also requires a 5% fetal bovine serum (FBS) supplementation and has a virus yield up to 3.1E7 plaque-forming units (PFU)/cm2. Using the animal-free cell culture medium VP-SFM™ and a new feeding strategy, we demonstrate a yield boost by a mean of 0.3 log while reducing seeding density to 5.0E3 cells/cm2 and cutting FBS supplementation by up to 40% during the production process. Additionally, FBS is completely removed at the time of harvest. Eleven commercial micro- and macrocarriers were screened regarding cell growth, bead-to-bead transfer capability, and virus yield. We present a proof-of-concept study for producing H-1PV on a large scale with the microcarrier Cytodex® 1 in suspension and a macrocarrier for a fixed-bed iCELLis® bioreactor. A carrier-based H-1PV production process combined with an optimized cell culture medium and feeding strategy can facilitate future upscaling to industrial-scale production. Key points • Virus yield increase and FBS-free harvest after switching to cell culture medium VP-SFM™. • We screened carriers for cell growth, bead-to-bead transfer capability, and H-1PV yield. • High virus yield is achieved with Cytodex® 1 and macrocarrier for iCellis® in Erlenmeyer flasks.

pUL36 de-ubiquitinase activity augments both the initiation and progression of lytic virus infection in IFN–primed cells

The conserved, structural HSV-1 tegument protein pUL36 is essential for both virus entry and assembly. While its N-terminal de-ubiquitinase (DUB) activity is dispensable for infection in cell culture, it is required for efficient virus spread in vivo by acting as a potent viral immune evasin. Here, we show that the pUL36 DUB activity was required to overcome interferon-(IFN)-mediated suppression of both plaque initiation and progression to productive infection. Immediately upon virus entry, incoming tegument-derived pUL36-DUB activity helped the virus to escape intrinsic antiviral resistance and efficiently initiate lytic virus replication in IFN-primed cells. Subsequently, de novo expressed pUL36-DUB augmented the efficiency of productive infection and virus yield. Interestingly, removal of IFN shortly after inoculation only resulted in a partial rescue of plaque formation, indicating that an IFN-induced defense mechanism eliminates invading virus particles unless counteracted by pUL36-DUB activity. Taken together, we demonstrated that the pUL36 DUB disarms IFN-induced antiviral responses at two levels, namely, to protect the infectivity of invading virus as well as to augment productive virus replication in IFN-primed cells.Author SummaryHSV-1 is an ubiquitous human pathogen that is responsible for common cold sores but may also cause life-threatening disease. pUL36 is an essential and conserved protein of infectious herpesvirus virions with a unique de-ubiquitinating (DUB) activity. The pUL36 DUB is dispensable for efficient virus infection in cell culture but represents an important viral immune evasin in vivo. Here, we showed that tegument-derived DUB activity delivered by the invading virus particles is required to overcome IFN-induced host resistance and to initiate efficient lytic infection. De novo expressed pUL36 DUB subsequently augments productive infection and virus yield. These data indicate that the pUL36 DUB antagonizes the activity of yet unidentified IFN-inducible E3 ligases to facilitate productive infection at multiple levels. Our findings underscore the therapeutic potential of targeting conserved herpesvirus DUBs to prevent or treat herpesvirus disease.

Adeno-associated Virus (AAV) Capsid Chimeras with Enhanced Infectivity Reveal a Core Element in the AAV Genome Critical for both Cell Transduction and Capsid Assembly

Adeno-associated viruses (AAV) have attracted significant attention in the field of gene and cell therapy due to highly effective delivery of therapeutic genes into human cells. The ability to generate recombinant AAV vectors compromised of unique or substituted protein sequences has led to the development of capsid variants with improved therapeutic properties. Seeking novel AAV vectors capable of enhanced transduction for therapeutic applications, we have developed a series of unique capsid variants termed AAV X-Vivo (AAV-XV) derived from chimeras of AAV12 VP1/2 sequences and the VP3 sequence of AAV6. These AAV variants showed enhanced infection of human primary T cells, hematopoietic stem cells, and neuronal cell lines over wildtype parental viruses, and superiority over AAV6 for genomic integration of DNA sequences by AAV alone or in combination with CRISPR gene editing. AAV-XV variants demonstrate transduction efficiency equivalent to AAV6 at multiplicities of infection 2 logs lower, enabling T cell engineering at low AAV doses. The protein coding sequence of these novel AAV chimeras revealed disruptions within the assembly-activating protein (AAP) which likely accounted for observed lower virus yield. A series of genome alterations, reverting the AAP sequence back to wildtype AAV6, had a negative impact on the enhanced transduction seen with AAV-VX, indicating overlapping functions within this sequence for both viral assembly and effective T cell transduction. Our findings show these AAV-XV variants are highly efficient at cell transduction at low dose and demonstrates the importance of the AAP coding region in both viral particle assembly and cell infection. IMPORTANCE A major hurdle to the therapeutic potential of AAV in gene therapy lies in achieving clinically meaningful AAV doses, and secondarily, ability to manufacture commercially viable titers of AAV to support this. By virtue of neutralizing antibodies against AAV that impede patient repeat-dosing, the dose of AAV for in vivo gene delivery has been high, which has resulted in unfortunate recent safety concerns and deaths in patients given higher-dose AAV gene therapy. We have generated new AAV variants possessing unique combinations of capsid proteins for gene and cell therapy applications termed AAV-XV, which have high levels of cell transduction and gene delivery at lower MOI. Furthermore, we demonstrate a novel finding, and an important consideration for recombinant AAV design, that a region of the AAV genome encoding the capsid viral protein and AAP is critical for both virus yield and the enhancement of infection/transduction.

Highly efficient production of an influenza H9N2 vaccine using MDCK suspension cells

The use of H9N2 subtype avian influenza vaccines is an effective approach for the control of the virus spread among the poultry and for the upgrading of vaccine manufacturing cell culture-based production platform could overcome the limitations of conventional egg-based platform and alternate it. The development of serum-free suspension cell culture could allow even higher virus productivity, where a suspension cell line with good performance and proper culture strategies are required. In this work, an adherent Mardin-Darby canine kidney (MDCK) cell line was adapted to suspension growth to cell concentration up to 12 × 106 cells/mL in a serum-free medium in batch cultures. Subsequently, the H9N2 influenza virus propagation in this MDCK cell line was evaluated with the optimization of infection conditions in terms of MOI and cell concentration for infection. Furthermore, various feed strategies were tested in the infection phase for improved virus titer and a maximum hemagglutinin titer of 13 log2 (HAU/50 μL) was obtained using the 1:2 medium dilution strategy. The evaluation of MDCK cell growth and H9N2 virus production in bioreactors with optimized operating conditions showed comparable cell performance and virus yield compared to shake flasks, with a high cell-specific virus yield above 13000 virions/cell. With the purified H9N2 virus harvested from the bioreactors, the MDCK cell-derived vaccine was able to induce high titers of neutralizing antibodies in chickens. Overall, the results demonstrate the promising application of the highly efficient MDCK cell-based production platform for the avian influenza vaccine manufacturing.

Highly efficient production of an influenza H9N2 vaccine using MDCK suspension cells

The use of H9N2 subtype avian influenza vaccines is an effective approach for the control of the virus spread among the poultry, and for the upgrading of vaccine manufacturing, cell culture-based production platform could overcome the limitations of conventional egg-based platform and alternate it. The development of serum-free suspension cell culture could allow even higher virus productivity, where a suspension cell line with good performance and proper culture strategies are required. In this work, an adherent Mardin–Darby canine kidney (MDCK) cell line was adapted to suspension growth to cell concentration up to 12 × 106 cells/mL in a serum-free medium in batch cultures. Subsequently, the H9N2 influenza virus propagation in this MDCK cell line was evaluated with the optimization of infection conditions in terms of MOI and cell concentration for infection. Furthermore, various feed strategies were tested in the infection phase for improved virus titer and a maximum hemagglutinin titer of 13 log2 (HAU/50 μL) was obtained using the 1:2 medium dilution strategy. The evaluation of MDCK cell growth and H9N2 virus production in bioreactors with optimized operating conditions showed comparable cell performance and virus yield compared to shake flasks, with a high cell-specific virus yield above 13,000 virions/cell. With the purified H9N2 virus harvested from the bioreactors, the MDCK cell-derived vaccine was able to induce high titers of neutralizing antibodies in chickens. Overall, the results demonstrate the promising application of the highly efficient MDCK cell-based production platform for the avian influenza vaccine manufacturing.

Triple Combination Nitazoxanide, Ribavirin, and Hydroxychloroquine results in the multiplicative reduction of in vitro SARS-CoV-2 viral replication

BackgroundAn immediate unmet medical need exists to test and develop existing approved drugs against SARS-COV-2. Despite many efforts, very little progress has been made regarding finding low-cost oral medicines that can be made widely available worldwide to address the global pandemic.MethodsWe sought to examine if a triple combination of nitazoxanide (using its active metabolite tizoxanide), ribavirin, and hydroxychloroquine would lead to a multiplicative effects on viral replication of SARS-COV-2 resulting in a significant reduction of virus yield using VERO E6 cells as a model of viral replication.ResultsVirus yield measured in PFU/ml was ~ 2 logs lower with triple combination versus either drug alone, resulting in the prolongation of time to peak cytopathic effects (CPE). The time to produce 50% CPE increased from 2.8 days for viral controls versus 5.3 days for triple combination therapy. Finally, for each 1-log reduction in virus yield 24 hours post-infection, there was an additional 0.7-day delay in onset of CPE.ConclusionsA triple combination of tizoxanide, ribavirin, and hydroxychloroquine produced a reduction in SARS-COV-2 viral replication in Vero E6 cells, warranting exploration in additional cell lines as well as human clinical trials.

A novel live attenuated RSV vaccine candidate with mutations in the L protein SAM binding site and the G protein cleavage site is protective in cotton rats and a rhesus macaque

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in children < 5 years of age worldwide, infecting the majority of infants in their first year of life. Despite the widespread impact of this virus, no vaccine is currently available. For over 50 years, live attenuated vaccines (LAV) have been shown to protect against other childhood viral infections, offering the advantage of presenting all viral proteins to the immune system for stimulation of both B and T cell responses and memory. The RSV LAV candidate described here, rgRSV-L(G1857A)-G(L208A), contains two modifications: an attenuating mutation in the S-adenosylmethionine (SAM) binding site of the viral mRNA cap methyltransferase (MTase) within the large (L) polymerase protein and a mutation in the attachment (G) glycoprotein that inhibits its cleavage during production in Vero cells, resulting in virus with a “non-cleaved G” (ncG). RSV virions containing the ncG have an increased ability to infect primary well-differentiated human bronchial epithelial (HBE) cultures which model the in vivo site of immunization, the ciliated airway epithelium. This RSV LAV candidate is produced efficiently in Vero cells, is highly attenuated in HBE cultures, efficiently induces neutralizing antibodies that are long-lasting, and provides protection against an RSV challenge in the cotton rat, without causing enhanced disease. Similar results were obtained in a rhesus macaque. Importance Globally, RSV is a major cause of death in children under one year of age, yet no vaccine is available. We have generated a novel RSV live attenuated vaccine candidate containing mutations in the L and G proteins. The L polymerase mutation does not inhibit virus yield in Vero cells, the cell type required for vaccine production, but greatly reduces virus spread in HBE cultures, a logical in vitro predictor of in vivo attenuation. The G attachment protein mutation reduces its cleavage in Vero cells, thereby increasing vaccine virus yield, making vaccine production more economical. In cotton rats, this RSV vaccine candidate is highly attenuated at a dose of 105 PFU and completely protective following immunization with 500 PFU, 200-fold less than the dose usually used in such studies. It also induced long-lasting antibodies in cotton rats and protected a rhesus macaque from RSV challenge. This mutant virus is an excellent RSV live attenuated vaccine candidate.

AAV Capsid Chimeras with Enhanced Infectivity reveal a core element in the AAV Genome critical for both Cell Transduction and Capsid Assembly

ABSTRACTAdeno-associated viruses (AAV) have attracted significant attention in the field of gene and cell therapy due to highly effective delivery of therapeutic genes into human cells. The ability to generate recombinant AAV vectors compromised of unique or substituted protein sequences has led to the development of capsid variants with improved therapeutic properties. Seeking a novel AAV capable of enhanced transduction of human T cells for applications in immunotherapy, we have developed a unique capsid variant termed AAV X-Vivo (AAV-XV) that is a chimera of AAV12 VP1/2 sequences and the VP3 sequence of AAV6. This AAV chimera showed enhanced infection of human primary T cells and hematopoietic stem cells, and superiority over wildtype AAV6 for the genomic integration of DNA sequences either by AAV alone or in combination with CRISPR gene editing. AAV-XV demonstrated transduction efficiency equivalent to AAV6 at multiplicities of infection 2 logs lower, enabling T cell engineering at low AAV doses. Analyzing the protein coding sequence of AAV-XV revealed disruptions within the assembly-activating protein (AAP) which likely accounted for observed lower virus yield. A series of genome alterations reverting the AAP sequence back to wildtype had a negative impact on the enhanced transduction seen with AAV-VX, indicating overlapping functions within this sequence for both viral assembly and effective T cell transduction. Our findings show that AAV-XV is highly efficient at T cell engineering at low AAV dose and demonstrates the importance of AAP coding region in both viral particle assembly and cell infection.IMPORTANCEA major hurdle to the therapeutic potential of AAV in gene therapy lies in achieving clinically meaningful AAV doses, and secondarily, ability to manufacture commercially viable titers of AAV to support this. By virtue of neutralizing antibodies against AAV that impede patient repeat-dosing, the dose of AAV for in vivo gene delivery has been high, which has resulted in unfortunate recent safety concerns and deaths in patients given higher-dose AAV gene therapy. We have generated a new AAV variant possessing a unique combination of capsid proteins for ex-vivo application termed AAV-XV, which delivers high levels of cell transduction and gene delivery at a lower MOI. Furthermore, we demonstrate a novel finding, and an important consideration for recombinant AAV design, that a region of the AAV genome encoding the capsid protein and AAP gene is critical for both virus yield and the enhancement of infection/transduction.

Cell-Based Influenza A/H1N1pdm09 Vaccine Viruses Containing Chimeric Hemagglutinin with Improved Membrane Fusion Ability

The H1N1 influenza pandemic vaccine has been developed from the A/California/07/09 (Cal) virus and the well-known high-yield A/Puerto Rico/8/34 (PR8) virus by classical reassortment and reverse genetics (RG) in eggs. Previous studies have suggested that Cal-derived chimeric hemagglutinin (HA) and neuraminidase (NA) improve virus yields. However, the cell-based vaccine of the H1N1 pandemic virus has been less investigated. RG viruses that contained Cal-derived chimeric HA and NA could be rescued in Madin–Darby canine kidney cells that expressed α2,6-sialyltransferase (MDCK-SIAT1). The viral growth kinetics and chimeric HA and NA properties were analyzed. We attempted to generate various RG viruses that contained Cal-derived chimeric HA and NA, but half of them could not be rescued in MDCK-SIAT1 cells. When both the 3′- and 5′-terminal regions of Cal HA viral RNA were replaced with the corresponding regions of PR8 HA, the RG viruses were rescued. Our results were largely consistent with those of previous studies, in which the N- and C-terminal chimeric HA slightly improved virus yield. Importantly, the chimeric HA, compared to Cal HA, showed cell fusion ability at a broader pH range, likely due to amino acid substitutions in the transmembrane region of HA. The rescued RG virus with high virus yield harbored the chimeric HA capable of cell fusion at a broader range of pH.