An Introduction for virus

1. Virus-Like Particles Based Vaccines Virus-Like Particles (VLPs) Based Vaccine, an approach that fights viruses with its own weapon, is one of the most exciting emerging vaccine technologies for generating effective and long-lasting protection. Creative Biolabs jumps on the bandwagon and has focused on VLP-based vaccine development for many years. Virus-like particles resemble and mimic the structure of real viruses without being infectious. Since they do not contain any viral genetic material, they have significant potential to trigger a strong immune response without causing any actual harm. Expression of viral structural proteins such as Envelope or Capsid can result in the self-assembly of VLPs. VLPs have been generated from components of various virus families including Retroviridae (e.g. HIV), Parvoviridae (e.g. adeno-associated virus), bacteriophages (e.g. Qβ, AP205) and Flaviviridae (e.g. Hepatitis C virus). VLP production may take advantage of several established systems, including bacterial, yeast, insect and mammalian cells. Types of Virus-like Particles Virus-like particles consist of one or more structural proteins that, when expressed recombinantly, have the ability to self-assemble. Proteins can be arranged in single, double or triple layers. The VLPs of human papilloma virus (HPV) are formed by a single structural protein that forms the basic capsid of the particle. Other more complex VLPs contain several structural proteins. For example, the VLPs of the Reoviridae family are formed by two to four different proteins arranged in several layers. VLPs may have an external lipid envelope such as HIV-1 VLPs. Influenza virus VLPs are also formed by the protein core and hemagglutinin spikes which are displayed on its surface. [1] Since envelope-VLPs will contain proteins expressed on their membranes, the choice of producer cell line will be crucial.

2. Fig 1. Different virus-like particles. [2] Creative Biolabs also provide the development of Chimeric VLP based vaccines. The chimeric VLPs consist of viral proteins while envelope proteins are from the second virus. The envelope protein can serve as a signal for a particular tissue receptor so that VLP can be targeted to a particular tissue with the capsid protein conjugated with the targeting component. Chimeric VLPs paved the way for the development of vaccine candidates with a broader, more powerful and comprehensive protection against diseases. Production Methods Creative Biolabs provides VLP production using various expression systems covering bacterial, yeast, insect mammalian cells and more. The choice of production systems depends on several factors, including cost and the need for post-translational modifications (PTMs), which is important in generating an optimal immune response. Table 1. Advantages and disadvantages of the different VLP production methods. [1] Production method Production method Advantages Advantages Disadvantages Disadvantages E. coli E. coli • Ease of expression • Ability to scale-up • Does not allow for glycosylation.

3. • Low production cost • Endotoxins Yeast • Ease of expression • Ability to scale-up • Low production cost • Non-appropriate protein glycosylation (i.e. high mannose glycoprotein modification). • Risk of incorrect folding & assembly. Insect cells • Can produce large amounts of correctly folded VLP in high density cell culture conditions • Ability to scale-up • The risk of culturing opportunistic pathogens is minimized compared to mammalian cell culture • Host-derived insect cell/baculovirus components may act as vaccine adjuvants, help trigger a more effective immune response • Limited to high mannose glycoprotein modification. • Baculovirus contaminants may be difficult to remove • Host-derived insect cell/baculovirus components may also mask the immune response against the desired epitope Mammalian cells • Producer cells more closely related to the natural host • Appropriate PTMs and authentic assembly of VLPs • Higher production cost • Lower productivities Plants • Ease of expression • Ability to scale-up • No human-derived virus contamination • Cannot undergo PTMs and VLP assembly • Low expression levels • Stability: antigen degradation Culture Modes Creative Biolabs offers our clients different culture modes for VLP production: batch, fed-batch, continuous or perfusion mode. Batch mode: the most used culture mode and has been used for many types of VLP, including HIV, Chikungunya virus, and Ebola virus. All the elements needed are added at the beginning of the culture with the advantage that the medium is well utilized, and the product is highly concentrated. Fed-batch mode: small quantities of nutrients or medium are added during the culture to supply the cells with specific components depleted. Such strategies can extend the exponential growth phase and be used to reach high cell concentrations. Continuous cultivation mode: fresh medium is added while the conditioned medium is extracted. This system presents a short “turn-around” time (reduces the cost associated with cleaning and filling of the bioreactor) and a small number of production steps.   

4. However, continuous production requires large amounts of medium, which is hard to adapted to large-scale production. Perfusion mode: large volumes of media and cell-medium separation devices are needed. Although enhancing cellular growth and protein expression, the perfusion system impact negatively on cellular growth rate, protein expression and production cost.  VLP-based Vaccines VLPs exhibit more similar conformational epitopes to the native viruses compared with individual proteins or peptides, and therefore antibody reactivity or immune system response is significantly improved. Most importantly, since VLPs can not replicate, they provide a safer choice for attenuated viruses. VLPs have been used to develop FDA-approved vaccines against hepatitis B and human papilloma virus. Several other VLP-based vaccine candidates are undergoing clinical trials or preclinical assessments, such as parvovirus, Norwalk, influenza and various chimeric VLPs. Research shows that VLP vaccines against influenza virus can provide stronger and longer-lasting protection against influenza viruses than conventional vaccines. Also, VLP vaccine production can start immediately (12 weeks) after the strain is sequenced while a traditional vaccine usually needs 9 months. Our Services Different types of VLPs for vaccine development: VLPs of structurally simple viruses (such as hepatitis E virus, HPV, norwalk virus, canine and porcine parvovirus VLPs); VLPs with lipid envelope (such as HIV, RSV, HBsAg, herpes simplex, NDV, HCV, Ebola, RVFV, Hantaan and Marburg virus VLPs); VLPs with multiple protein layers (such as IBDV, poliovirus, bluetongue virus, enterovirus 71 and rotavirus VLPs); A range of VLP production systems cover bacterial, yeast, insect mammalian cells and more; Different culture modes for VLP production: batch, fed-batch, continuous or perfusion mode; Downstream processing of VLPs; Quality monitor & control.      In the process of VLP based vaccines design, development, and manufacturing, working with partners who understand the challenges can dramatically increase efficiency. Creative Biolabs' proven application experience can help you overcome the challenges in the VLP vaccines development process. References

5. 1.Fuenmayor J; et al. Production of virus-like particles for vaccines. N Biotechnol. 2017, 39(Pt B): 174-180. 2.Chroboczek J; et al. Virus-like particles as vaccine. Acta Biochim Pol. 2014, 61(3): 531-539.