Characterization of a quail suspension cell line for production of a fusogenic oncolytic virus.

The development of efficient processes for the production of oncolytic viruses (OV) plays a crucial role regarding the clinical success of virotherapy. Although many different OV platforms are currently under investigation, manufacturing of such viruses still mainly relies on static adherent cell cultures, which bear many challenges, particularly for fusogenic OVs. Availability of GMP-compliant continuous cell lines is limited, further complicating the development of commercially viable products. BHK21, AGE1. CR and HEK293 cells were previously identified as possible cell substrates for the recombinant vesicular stomatitis virus (rVSV)-based fusogenic OV, rVSV-NDV. Now, another promising cell substrate was identified, the CCX.E10 cell line, developed by Nuvonis Technologies. This suspension cell line is considered non-GMO as no foreign genes or viral sequences were used for its development. The CCX.E10 cells were thus thoroughly investigated as a potential candidate for OV production. Cell growth in the chemically defined medium in suspension resulted in concentrations up to 8.9 × 106 cells/mL with a doubling time of 26.6 h in batch mode. Cultivation and production of rVSV-NDV, was demonstrated successfully for various cultivation systems (ambr15, shake flask, stirred tank reactor, and orbitally shaken bioreactor) at vessel scales ranging from 15 mL to 10 L. High infectious virus titers of up to 4.2 × 108 TCID50 /mL were reached in orbitally shaken bioreactors and stirred tank reactors in batch mode, respectively. Our results suggest that CCX.E10 cells are a very promising option for industrial production of OVs, particularly for fusogenic VSV-based constructs.

A whole virus pandemic influenza H1N1 vaccine is highly immunogenic and protective in active immunization and passive protection mouse models.

The recent emergence and rapid spread of a novel swine-derived H1N1 influenza virus has resulted in the first influenza pandemic of this century. Monovalent vaccines have undergone preclinical and clinical development prior to initiation of mass immunization campaigns. We have carried out a series of immunogenicity and protection studies following active immunization of mice, which indicate that a whole virus, nonadjuvanted vaccine is immunogenic at low doses and protects against live virus challenge. The immunogenicity in this model was comparable to that of a whole virus H5N1 vaccine, which had previously been demonstrated to induce high levels of seroprotection in clinical studies. The efficacy of the H1N1 pandemic vaccine in protecting against live virus challenge was also seen to be equivalent to that of the H5N1 vaccine. The protective efficacy of the H1N1 vaccine was also confirmed using a severe combined immunodeficient (SCID) mouse model. It was demonstrated that mouse and guinea pig immune sera elicited following active H1N1 vaccination resulted in 100% protection of SCID mice following passive transfer of immune sera and lethal challenge. The immune responses to a whole virus pandemic H1N1 and a split seasonal H1N1 vaccine were also compared in this study. It was demonstrated that the whole virus vaccine induced a balanced Th-1 and Th-2 response in mice, whereas the split vaccine induced mainly a Th-2 response and only minimal levels of Th-1 responses. These data supported the initiation of clinical studies with the same low doses of whole virus vaccine that had previously been demonstrated to be immunogenic in clinical studies with a whole virus H5N1 vaccine.

Development of a live-attenuated influenza B DeltaNS1 intranasal vaccine candidate.

We discovered a unique, single amino acid mutation in the influenza B M1 protein promoting viral growth of NS1 truncation mutants in Vero cells. Due to this mutation, we were able to generate an influenza B virus lacking the complete NS1 open reading frame (DeltaNS1-B virus) by reverse genetics, which was growing to titers of 8log(10)TCID(50)/ml in a Vero cell culture-based micro-carrier fermenter. The DeltaNS1-B vaccine candidate was attenuated in IFN-competent hosts such as human alveolar epithelial cells (A549) similar to influenza A DeltaNS1 viruses. In ferrets, the DeltaNS1-B virus was replication-deficient and did not provoke any clinical symptoms. Importantly, a single intranasal immunization of ferrets at a dose as low as 6 log(10)TCID(50)/animal induced a significant HAI response and provided protection against challenge with wild-type influenza B virus. So far, the lack of a DeltaNS1-B virus component growing to high titers in cell culture has been limiting the possibility to formulate a trivalent vaccine based on deletion of the NS1 gene. Our study closes this gap and paves the way for the clinical evaluation of a seasonal, trivalent, live replication-deficient DeltaNS1 intranasal influenza vaccine.

Emerging trends in plasma-free manufacturing of recombinant protein therapeutics expressed in mammalian cells.

Mammalian cells are the expression system of choice for therapeutic proteins, especially those requiring complex post-translational modifications. Traditionally, these cells are grown in medium supplemented with serum and other animal- or human-derived components to support viability and productivity. Such proteins are also typically added as excipients and stabilizers in the final drug formulation. However, the transmission of hepatitis B in the 1970s and of hepatitis C and HIV in the 1980s through plasma-derived factor VIII concentrates had catastrophic consequences for hemophilia patients. Thus, due to regulatory concerns about the inherent potential for transmission of infectious agents as well as the heterogeneity and lack of reliability of the serum supply, a trend has emerged to eliminate the use of plasma-derived additives in the production and formulation of recombinant protein therapeutics. This practice began with products used in the treatment of hemophilia and is progressively expanding throughout the entire industry. The plasma-free method of producing recombinant therapeutics is accomplished by the use of both cell culture media and final product formulations that do not contain animal- or human-derived additives. A number of recombinant therapeutic proteins for the treatment of several different diseases have been produced by plasma-free processes, with the objective of improving safety by eliminating blood-borne pathogens or by reducing immunogenicity. This review describes the factors that drove the development of plasma-free protein therapeutics and provides examples of advances in manufacturing that have made possible the removal of human and animal-derived products from all steps of recombinant protein production.

Cell culture (Vero) derived whole virus (H5N1) vaccine based on wild-type virus strain induces cross-protective immune responses.

The rapid spread and the transmission to humans of avian influenza virus (H5N1) have induced world-wide fears of a new pandemic and raised concerns over the ability of standard influenza vaccine production methods to rapidly supply sufficient amounts of an effective vaccine. We report here on a robust and flexible strategy which uses wild-type virus grown in a continuous cell culture (Vero) system to produce an inactivated whole virus vaccine. Candidate vaccines based on clade 1 and clade 2 influenza H5N1 strains were developed and demonstrated to be highly immunogenic in animal models. The vaccines induce cross-neutralising antibodies, highly cross-reactive T-cell responses and are protective in a mouse challenge model not only against the homologous virus but also against other H5N1 strains, including those from another clade. These data indicate that cell culture-grown whole virus vaccines, based on the wild-type virus, allow the rapid high yield production of a candidate pandemic vaccine.

The preclinical testing of a formaldehyde inactivated Ross River virus vaccine designed for use in humans.

Ross River virus was grown in industrial facilities in vaccine-certified Vero cells in the absence of serum, inactivated using standard formalin-inactivation protocols, treated with Benzonase to digest host cell DNA and purified on a sucrose gradient. Mice given two subcutaneous injections of 0.625 microg of this vaccine or two doses of 0.156 microg vaccine with aluminium hydroxide adjuvant failed to develop a detectable viraemia after intravenous challenge with 10(6)TCID50 of the prototype strain of Ross River virus (T48). Guinea pigs immunised with one or two10 microg doses of vaccine with adjuvant also failed to develop a detectable viraemia following a similar challenge. The levels of neutralising antibody (neutralisation index 1.9-3.1) in the mice protected against challenge with 10(6)TCID50 Ross River virus were similar to those in 16 former epidemic polyarthritis patients (1.1-3.5) who had not experienced a second clinical infection with Ross River virus in the 20 years following their initial infection.

A double-inactivated whole virus candidate SARS coronavirus vaccine stimulates neutralising and protective antibody responses.

A double-inactivated, candidate whole virus vaccine against severe acute respiratory syndrome associated coronavirus (SARS-CoV) was developed and manufactured at large scale using fermenter cultures of serum protein free Vero cells. A two step inactivation procedure involving sequential formaldehyde and U.V. inactivation was utilised in order to ensure an extremely high safety margin with respect to residual infectivity. The immunogenicity of this double-inactivated vaccine was characterised in the mouse model. Mice that were immunised twice with the candidate SARS-CoV vaccine developed high antibody titres against the SARS-CoV spike protein and high levels of neutralising antibodies. The use of the adjuvant Al(OH)3 had only a minor effect on the immunogenicity of the vaccine. In addition, cell mediated immunity as measured by interferon-gamma and interleukin-4 stimulation, was elicited by vaccination. Moreover, the vaccine confers protective immunity as demonstrated by prevention of SARS-CoV replication in the respiratory tract of mice after intranasal challenge with SARS-CoV. Protection of mice was correlated to antibody titre against the SARS-CoV S protein and neutralising antibody titre.