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Live-Attenuated Influenza Vaccine

Polymun has established a nasal spray vaccine employing live attenuated influenza strains we call Vero-Vac. It is produced with our proprietary, animal-component free cell culture technology using the Vero cell line. In addition, Influenza virus is an excellent carrier for foreign epitopes. At Polymun vaccine candidates were constructed for HIV and tuberculosis (TB).

 

The concept of attenuating influenza strains at low temperature was carried out using Vero cell culture instead of fertilized hen eggs. In serial passages of both influenza A and B viruses in Vero cells at 25°C several mutations were introduced leading to our donor or master strains. Subsequently, the vaccine strains are created by genetic reassortment of the two genes coding for the surface antigens hemagglutinin and neuraminidase from the contemporary wild type strain and all other genes from the cold adapted, attenuated proprietary master strain. Due to this composition, the vaccine strains acquire the attenuation with the temperature sensitive and cold adapted phenotype from the master strain, but at the same time display the antigenic properties of the contemporary pathogenic virus strain.

The whole process of creating and producing the strains is done using the Vero cell line. This African green monkey kidney cell line has already been accepted for the production of other live and inactivated vaccines. Polymun has developed a completely defined, animal-component-free cultivation system, thus providing additional safety in combination with excellent economy. The use of a continuous primate cell line does not only improve safety and scalability of the production process, it also generates viruses that reflect their epidemic counterparts better than egg produced strains.

The live attenuated influenza vaccine is administered intranasally as a spray thus mimicking the natural way of infection. A clinical trial comparing it to the successful Russian vaccine of the similar type (produced in fertilized hen eggs) was performed with encouraging results.

In addition, a second generation of influenza vaccines was developed at Polymun. It is based on the attenuation of influenza viruses by engineering the non-structural (NS) gene. This approach creates a new type of live attenuated influenza vaccines and viral vectors. We demonstrated the possibility to modulate influenza virus pathogenicity by truncation of the NS1 protein in mice. Moreover, hyperattenuated and replication-deficient NS mutants used as intranasal vaccines showed the same protection against the virulent virus challenge in the same way as normally replicating vaccines. The truncated NS1 proteins stimulate the immune response by inducing the release of interferon, pro-inflammatory cytokines and chemokines.

The NS gene technology not only can provide an interesting alternative to the current approaches for live influenza vaccines, they can also function as carrier of foreign antigens in the NS1 reading frame. For that purpose, Polymun has established functional techniques and selection systems (using the interferon deficient Vero cell line) to engineer and to introduce large foreign gene sequences into the NS1 gene of influenza. As a model we inserted a functional green fluorescence protein (GFP).

HIV

Based on our technology Polymun has engineered various HIV-gene segments into its flu-vectors and used them for nasal spray immunization. Promising cellular and humoral immune response in the murine model was obtained.

TB

The ESAT-6 antigen from mycobacterium tuberculosis was introduced into the NS1 gene of influenza A. In the mouse model these vaccine candidates were compared with the currently used BCG vaccine. Challenging the animals with the mouse pathogenic strain mycobacterium bovis displayed significantly improved protection compared to the BCG vaccine.

Polymun is looking for an investor who wants to continue with this technology.

PATENTS

Live vaccine and method of manufacture
granted by: AU 2002223560; CA 2,423,038; CN ZL01819206.8; EP 1 358 319; JP 5063852; KR 0927517; US 7,494,659

Recombinant influenza A viruses
granted by: AU 2001250352; EP 1 259 629; RU 2280690; US 6,800,288

PUBLICATIONS

Lohr V, Genzel Y, Jordan I, Katinger D, Mahr S, Sandig V, Reichl U (2012) Live attenuated influenza viruses produced in a suspension process with avian AGE1.CR.pIX cells. BMC Biotechnol 12:79

Ferko B, Kittel C, Romanova J, Sereinig S, Katinger H, Egorov A (2006) Live attenuated influenza virus expressing human interleukin-2 reveals increased immunogenic potential in young and aged hosts. J Virol 80(23):11621-7

Sereinig S, Stukova M, Zabolotnyh N, Ferko B, Kittel C, Romanova J, Vinogradova T, Katinger H, Kiselev O, Egorov A (2006) Influenza virus NS vectors expressing the mycobacterium tuberculosis ESAT-6 protein induce CD4+ Th1 immune response and protect animals against tuberculosis challenge. Clin Vaccine Immunol 13(8):898-904

Stukova MA, Sereinig S, Zabolotnyh NV, Ferko B, Kittel C, Romanova J, Vinogradova TI, Katinger H, Kiselev OI, Egorov A (2006) Vaccine potential of influenza vectors expressing Mycobacterium tuberculosis ESAT-6 protein. Tuberculosis 86(3-4):236-46

Kittel C, Ferko B, Kurz M, Voglauer R, Sereinig S, Romanova J, Stiegler G, Katinger H, Egorov A (2005) Generation of an influenza A virus vector expressing biologically active human interleukin-2 from the NS gene segment. J Virol 79(16):10672-7

Stasakova J, Ferko B, Kittel C, Sereinig S, Romanova J, Katinger H, Egorov A (2005) Influenza A mutant viruses with altered NS1 protein function provoke caspase-1 activation in primary human macrophages, resulting in fast apoptosis and release of high levels of interleukins 1beta and 18. J Gen Virol 86(1):185-95

Ferko B, Stasakova J, Romanova J, Kittel C, Sereinig S, Katinger H, Egorov A (2004) Immunogenicity and protection efficacy of replication-deficient influenza A viruses with altered NS1 genes. J Virol 78(23):13037-45

Kittel C, Sereinig S, Ferko B, Stasakova J, Romanova J, Wolkerstorfer A, Katinger H, Egorov A (2004) Rescue of influenza virus expressing GFP from the NS1 reading frame. Virology 324(1):67-73

Romanova J, Katinger D, Ferko B, Vcelar B, Sereinig S, Kuznetsov O, Stukova M, Erofeeva M, Kiselev O, Katinger H, Egorov A (2004) Live cold-adapted influenza A vaccine produced in Vero cell line. Virus Res 103(1-2):187-93

Mochalova L, Gambaryan A, Romanova J, Tuzikov A, Chinarev A, Katinger D, Katinger H, Egorov A, Bovin N (2003) Receptor-binding properties of modern human influenza viruses primarily isolated in Vero and MDCK cells and chicken embryonated eggs. Virology 313(2):473-80

Poomputsa K, Kittel C, Egorov A, Ernst W, Grabherr R (2003) Generation of recombinant influenza virus using baculovirus delivery vector. J Virol Methods 110(1):111-4

Romanova J, Katinger D, Ferko B, Voglauer R, Mochalova L, Bovin N, Lim W, Katinger H, Egorov A (2003) Distinct host range of influenza H3N2 virus isolates in Vero and MDCK cells is determined by cell specific glycosylation pattern. Virology 307(1):90-7

Ferko B, Stasakova J, Sereinig S, Romanova J, Katinger D, Niebler B, Katinger H, Egorov A (2001) Hyperattenuated recombinant influenza A virus nonstructural-protein-encoding vectors induce human immunodeficiency virus type 1 Nef-specific systemic and mucosal immune responses in mice. J Virol 75(19):8899-908

Garcia-Sastre A, Egorov A, Matassov D, Brandt S, Levy DE, Durbin JE, Palese P, Muster T (1998) Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology 252(2):324-30

Ferko B, Katinger D, Grassauer A, Egorov A, Romanova J, Niebler B, Katinger H, Muster T (1998) Chimeric influenza virus replicating predominantly in the murine upper respiratory tract induces local immune responses against human immunodeficiency virus type 1 in the genital tract. J Infect Dis 178(5):1359-68

Egorov A, Brandt S, Sereinig S, Romanova J, Ferko B, Katinger D, Grassauer A, Alexandrova G, Katinger H, Muster T (1998) Transfectant influenza A viruses with long deletions in the NS1 protein grow efficiently in Vero cells. J Virol 72(8):6437-41