UNIVERSITY OF QUEENSLAND LIBRARY

Virus-like Particle
Literature Review
A broad outline into the scientific reasoning behind VLP development
and production with a greater emphasis on the production of the Human
papillomavirus vaccine.

Patrick Deane – S4213343

 Virus-like Particle Literature Review 2010

1. Contents
1. Contents..................................................................................................................................... 1
2. Table of Figures...................................................................................................................... 2
3. Introduction............................................................................................................................. 3
4. Review........................................................................................................................................ 5
4.1. Introduction.................................................................................................................................................... 5
4.2. Upstream Processing................................................................................................................................... 6
4.3. Downstream Processing............................................................................................................................. 9
4.4. Advantages.................................................................................................................................................... 11
4.5. Conclusions................................................................................................................................................... 12
5. Literature review examining the human papillomavirus production and the
improvements in screening techniques............................................................................... 14
5.1. Introduction.................................................................................................................................................. 14
5.2. Production of HPV vaccine...................................................................................................................... 15
5.3. Possible changes that can be made to the analysis methods used in VLP production...18
5.4. Conclusions................................................................................................................................................... 22
6. Conclusions............................................................................................................................ 24
7. References.............................................................................................................................. 27

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2. Table of Figures

Figure 1 - In vitro technique that can be used to simplify the expression of VLP's. .......................8
Figure 2 - HPV vector generation......................................................................................................16
Figure 3 - Electron microscopy of a VLP mixture without gel filtration.........................................17
Figure 4 - VLP mixture after gel filtration........................................................................................18
Figure 5 - Diagram showing the FFF technique...............................................................................19
Figure 6 - A diagram showing the AFFF technique where A shows the focussing, injection and
relaxation stages and B shows the elution stage.............................................................................21

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3. Introduction
Vaccinations are one of the most efficient methods for reducing the mortality rate

associated with viruses and bacteria. Once people are vaccinated against a disease they

have a far lower risk of being infected by the disease and as a result are unable to pass

the disease on to non-vaccinated people. The world health organisation (2009) has

stated that one of the main reasons for the decreased mortality rates has been the

introduction and acceptance of vaccinations across the world. The main requirement of

a vaccine is that it produces potent memory B and T cells in the human immune system.

A newer technology that is becoming being touted as the most successful replacement

of the current vaccination program is the virus like particle (VLP) vaccine. As the

traditional way of creating vaccines is a long and tedious process many different ideas

have been tested. In the last decade many candidate polypeptides have been identified

that are able to repel a variety of infectious agents. These though have to be delivered to

the immune system where it is able to recognise and destroy the infectious agent.

Peptides cannot be used however, as they are too small to be good immunogens and

therefore require a carrier to enhance their immunogenicity. (Chackerian

2007)Methods like chemical coupling to large proteins are used which involves a very

complicated process. Peptides have also been fused to genes coding for virus capsid

proteins allowing self assembly in the viral capsid. Although this does produce strong

antibody responses it does not induce the ‘T’ helper responses.

Instead of pathogens it has been found that viruses provide a far greater stimulant for

the induction of local and general secretary and systematic immune responses. As

viruses are very organised they are promote a far greater quality of response from the B

cell. There are two methods that can be used involving viruses as the delivery system.

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The first uses live viruses that replicate easily to promote an antibody response.

Because of the strong side effects that this can cause VLP’s are being found to help

reduce the potential side effects of the vaccine. The first expression of VLPs was

hepatitis B virus surface particles in the 1980s. (Noad, Roy, 2003) However, the viral

proteins where rarely expressed in the correct way because of its manufacture in yeast.

Therefore, VLP’s only started to have a future much later on when more powerful

expression systems like baculovirus expression systems could be utilized to express the

protein in the correct expression and its subsequent assembly of viral capsid proteins to

create new VLPs. (Casal, 2001)

VLP technology has allowed a number of newer vaccines to be produced most

noticeably for cervical cancer where VLP based vaccines have been created and sold

under the names Gardasil and Cervarix. (Noad, Roy, 2003) VLP technology is also being

used to help discover potential vaccines for many other diseases including influenza and

hepatitis C amongst others.

This report outlines the production methods of the VLP vaccine including the general

downstream and upstream processing commonly used in pharmaceutical companies.

The advantages of VLP technologies are also described showing why it is attracting also

much attention. It draws from information mainly gathered about the human

Papillomavirus (HPV) vaccine but other potential VLP vaccine productions have also

been considered. A literature review in Chapter 4 then shows how the HPV vaccine can

be produced in E coli and then considers a better analysis technique in Field-flow

fractionation and how this can help improve current manufacturing processes.

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4. Review
5. Introduction
Virus like particle (VLPs) vaccines comprise of multiple copies of a protein antigen that

when it is assembled together mimics the conformation of the native virus. Therefore,

VLPs resemble viruses, yet are non-infectious since they do not contain viral genetic

material that will infect the organism and further viral replication. (Lee, et al, 2009) The

first VLP was described thirty years ago using a technique using the Hepatitis B virus,

which was expressed in yeast. The human body responds to the VLP vaccine as if it is

seeing the live version of the virus allowing the body to build up an immune system

capable of fighting the live virus. Without the genetic material VLP vaccines are

incapable of causing the infections themselves while at the same time presenting the

viral antigens in the most authentic configuration possible.

Normally anti viral vaccines have traditional been prepared by using weakened forms of

the infectious virus. This type of vaccine although successful in many cases involves

inherent complications in the manufacturing process and has some limited applications

in the treatment of the population with many side effects being reported so VLP is seen

as an advantageous route due to its lack of viral DNA.

As already stated the main requirement of a vaccine is that it produces potent memory

B and T cells in the human immune system. To achieve this effect it has normally been

advantageous to vaccinate the host using the route used by the target microorganism.

The immune system has three lines of defences, which consist of a physical barrier, an

innate immune system and an adaptive immune system. The adaptive immune system

produces a lot of receptors of random specificity across the B and T cells. These cells are

created when lymphocytes originate and differentiate in the bone marrow creating B

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cells, or differentiate in the thymus to give T cells. (Chackerian 2007) B cells produces

antibodies, which function to neutralise viruses. When an antigen is recognised but the

receptors the B cell activates, maturing and selecting which antibody to produce. This is

followed by affinity maturation where B cells that have an increased infinity for the

specific antigen are used. These cells then can either become plasma cells that secrete

antibodies, or memory cells that allow the host to have long lasting protection against

an attack by the same microorganism. T cells provide assistance to the B cells with

antibody productions and therefore vaccines need to induce the appropriate B and T

cell responses.

The main way to produce VLP vaccine is when the capsid protein of the virus is

expressed in a cell culture where it spontaneously forms into VLPs, each comprised of

180 copies of the protein. Another type of virus have viral envelopes covering the

protein capsid shells for example herpes. The envelopes are normally derived from a

portion of the host cell membranes in a process called budding. During the budding

process newly formed virus particles become enveloped in the outer membrane

forming a viral envelope, and the capsid is this protein coat around the virus. The

unique structure of these enveloped viruses continues to pose problems to the

bimolecular engineers. The methods used to create VLPs have been shown to elicit

strong T cell and B cell immune responses.

6. Upstream Processing
Scientists in the early 1980’s found that the expression of viral structural proteins could

lead to the self assembly of regular structure VLPs with unique properties. (Maranga, et

al, 2004) Genes are used which express the protein used to form the structure of the

virus. These are then inserted into vectors where certain promoters allow them to be

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expressed in the host cell under a specific condition. One of the main obstacles to

efficient use of VLP technology is the production method. VLP’s are currently being

generated using expression hosts such as yeast, bacteria and mammalian cells. (Garcea,

Gissmann, 2004) There are many different advantages and disadvantages to each of

these different host cells. For example yeast cells allow large scale production and

simpler downstream processing; however have lower post translational modifications

then mammalian cells. The host cells are decided depending on the amount of VLPs

needed and the function of the vaccine amongst others. These host cells assemble the

structural protein in vivo therefore bypassing the need to understand the process fully

and eliminating the chance to implement control process. This type of assembly is

widely used throughout the VLP industry, most noticeably with the creation of human

papillomavirus vaccinations, where VLP vaccines where created using bacteria or insect

cells. However, this process does have a disadvantage, as the particles are assembled in

vivo a number of contaminates can be contained within the VLP requiring a number of

steps to remove them. These contaminated particles must also be isolated from the

VLPs created during the bioprocess. (Chuan, 2009)Therefore, this simple upstream

process can vastly complicate the downstream process. This in vivo assembly therefore

overcomplicates the method creating a flawed production as the underlying

methodology is not understood, making the process hard to scale up. New production

methods are being considered where the VLP’s are not created in cells rather in vitro.

An example of this has been proposed by Pattenden et al., (2005) allowing a much

greater control to be exercised over the process decreasing the amount of downstream

processing required. This can be seen in figure one where protein subunits are

manufactured using a recombinant expression system. These subunits are then

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extracted from the cells and purified and are then self assembled in a reactor using

controlled conditions.

Figure 1 - In vitro technique that can be used to simplify the expression of VLP's. (Chuan, 2009)
There are two steps in the bio-reaction to produce VLPs. The first of these is a growth

step where the cells, mammalian bacteria etc, are allowed to grow to a set boundary,

after this a baculovirus step occurs where the VLP production occurs. In the case of

VLPs production in the growth step is non existing and therefore the main boundary

condition is the cell concentration. This can be affected by the culture medium, the

reactor type and its conditions. The bioreactor type chosen is dependent on the host cell

and the final product needed. For example, with insect cells after the VLP has started to

be expressed, the cell grows in size making it more sensitive to stress and therefore

more liable to break. The bioreactor used therefore would need to have a stirrer which

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would have a lower shear stress increasing the products concentration. (Cruz, Maranga,

Carrondo, 2009)

7. Downstream Processing
The integration of the upstream and downstream processing have to be considered in

the overall plan for the production of any VLPs. As already stated using an in vivo bio-

process increases the downstream time required. Other factors also need to be

considered for example the medium used in the bioreactor. Certain compounds can be

added to increase the productivity if they are easily removed in the downstream

processing. The use of serum free mediums decreases the number of contaminants and

therefore the downstream time decreasing the cost of the production to the company.

There are three purification steps normally considered for VLP purification. (Maranga,

et al, 2004) The first of these is recovery which concerns the separation of the cells

from the fermentation mixture. There are two methods that can normally be used which

is centrifugation or filtration. The method used is dependent on the solid content;

however filtration is increasingly used as it does not bind the cell together. Within

filtration there are many different types that can be used, depending on the size of the

product, the amount of flow etc. As the VLP’s are inside the cell the next stage is

normally cell lysis allowing the VLP to separate from the cells content. This can be

achieved by using homogenization, bead mills, chemical or biological fluids amongst

others. Cell disruption generates a complex mixture containing cell debris amongst

other materials which is insoluble. The properties of the cell debris, in particular the

size, control the performance of further downstream processing. The next stage is

isolation where the contaminants are isolated, as are those that have vastly different

properties to that of the VLP. The main techniques used are precipitation and extraction

which both rely on a difference between the charges and solubility of the antigen and

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contaminants. Some techniques use enzymes to remove RNA/DNA, proteins and lipids

which constitute the major molecules of contaminant. (Casal, 2001) Further isolation

steps are then required to remove the enzymes from the recovered stream. Both

precipitation and isolation are currently used to concentrate the antigen for further

treatment in the purification phase. The last purification phase is to remove any

contaminates that may have properties that closely resemble that of the VLP thus

requiring more complicated techniques which rely on only very small differences

between the contaminant and VLP. Centrifugation is a popular method used with the

separation being based on density or sedimentation rates for example, although other

less common techniques can be used. In the final step chromatography is used to help

manufacture highly pure products of the VLPs. Many different chromatography

techniques are often used in the same process to create the desired result including, ion

exchange chromatography, hydrophobic-interaction chromatography, size-exclusion

chromatography etc. (Maranga, et al, 2004) The finished product is then placed in a fluid

form in containers awaiting testing.

The product is therefore produced in batch conditions and as the quality of the VLP’s

are dependent on the specific conditions that the VLP was created in invoking varying

responses from the immune system so each batch has to be individually tested to find

the purity of the sample. There are a wide range of techniques available including

electron microscopy, SPS Phage, size exclusion chromatography and newer techniques

such as field flow fractionation. (Casal, 2001) These techniques are employed to make

sure that the purity of the VLP is above a certain level and that there are no toxic

contaminants present in the fluid, allowing the manufacturing process to be changed if

there is a significant deviation from the standards set.

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8. Advantages
Virus like particles are becoming more prominent in the world’s attention due to

several high profile vaccines being created. The cause for this increase in scientific

interest is due to the potential advantages compared to using live viruses. The cervical

cancer vaccine which has been developed using VLP technology has successfully

vaccinated 100% of people in stage three trials, which is a near unheard of number in

vaccination testing. (Villa, 2006)This massive increase in successful vaccinations has

lead to companies looking for other vaccines that could be made using VLP technology.

An example of the advantages of VLPs is explained using the influenza virus. This virus

has quite a simple structure with a protein coat which is tipped with two major proteins

encasing a strand of RNA. This is used to replicate the virus which contains eight genes.

The variation of two proteins is what causes the deviation to other viruses, and is used

by scientists to classify the virus. In the past scientists have had to inject a seed virus

into chicken eggs. These are then allowed to incubate and the seed virus replicating

hundreds of times within each egg. The eggs are then harvested and purified, before

being inactivated with heat or chemicals. The fragments that are left over are used to

create vaccines. The whole process can take over six months.

Virus like particle technology though can take months of this timeframe. This is because

an insect cell can be injected with three of the eight influenza genes. One gene will

contain instructions on how to build the viral cell while the other two genes code for the

structure of the proteins on the outside of the shell. The output is a hollow viral cell that

does not contain any live viruses. The risks that can occur by using live viruses in the

process such as reversion and incomplete inactivation, major manufacturing concerns

for traditional vaccines, can thus be completely avoided. (Chackerian, 2007) This

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process is therefore far quicker as it does not rely on the Centre for Disease Control to

make a seed virus, instead it just needs to wait for that years predicted virus strain to be

published and the genes can then be synthesized and injected into the insect cells. The

main downside of VLP’s is that they have not been around long enough to measure the

potential side effects of the vaccines. (Eric Bland, 2009)

VLPs therefore have a number of advantages over traditional vaccines. Because they

more closely match an individual viral strain, VLPs can trigger a more robust immune

response. In addition, live viruses are not needed to produce a VLP vaccine. This new

class of vaccine offers unprecedented immune-protection, inherent safety, and a simple

route of administration as it does not require large repeated doses. (Chuan, 2009)

9. Conclusions
It has been shown by using various case studies that VLPs can be produced easily and

can be manufactured into vaccines. As the VLP has no viral DNA it does not harmfully

infect the host, instead it will allow humans to build up their immune response

including activating the B and T cell, allowing them to form active memories of the

supposed virus. This is because the body responds to the VLP vaccine as if it is a real

virus as the structure mimics the virus structure. VLP production mimics that of normal

bio-processing in as far that there are upstream and downstream process that follow

the generic structure of bio-processing. Genes that are used by the virus to express the

structure of the virus are expressed into vectors with the addition of promoters, etc.

Various host systems can be used to express the VLP vaccines including yeast and

mammalian cells and there are ongoing studies into the preeminent expression host for

different VLPs. The host is then allowed to grow in a bio-reactor where after a set

growth period will start to express the VLP vaccine under fixed conditions. The process

then enters the upstream processing where the VLP has to be isolated and purified from

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the cells and other contaminants. The first step needs to isolate the cells from the

medium usually by a centrifuge and then the cells are mechanically broken apart by

bead mills. The mixture then undergoes further refinement techniques to separate it

from the cells content and then from other contaminants that have the same shape by a

series of filtration steps using centrifugation, chromatography etc. VLP’s have been

shown to have various advantages over other vaccines including a decreased risk of side

effects and faster production of a vaccine from scratch, making them a popular

alternative to using live viruses.

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10. Literature review examining the human papillomavirus

production and the improvements in screening techniques.
11. Introduction
As stated in the previous chapter virus like particle have already been assembled

creating a vaccine for the Human Papillomavirus (HPV). This section will look into the

production and synthesis of this vaccine and look into ways into improving its screening

techniques using field flow fractionation. The two main cases that have been studied are

(Chuan, et al, 2008) and (Sasagawa, et al, 1995).

Cervical cancer has been recognised as being a result of HPV infection of the cervical

epithelium. HPV is the second most sexually transmitted disease and as a result cervical

cancer is the second most common cancer in woman worldwide with over 250,000

deaths occurring each year. (Schiller, 1999) Common techniques that were used before

HPV vaccines became widespread included pap smears which helped reduce the

mortality rate of the cancer. There are over 100 types of HPV genomes with the highest

risk genotypes being HPV types 16 and 18 which are responsible for over 70% of

cervical cancer infections. (Silva, et al, 2001). Although HPV causes cervical cancer it

only occurs in less than five percent of infections and after a period of 20 years

following the initial infection.

There are two main types of vaccinations that can be created, prophylactic or

therapeutic. As stated in Moyle et al (2005) prophylactic vaccines are designed to

prevent HPV infection through induction of neutralising antibodies. In comparison,

therapeutic vaccines are designed to eradicate HPV infected cells by eliciting memory

CD8+ cells specific for HPV infected cells. So far only Prophylactic vaccinations are

currently available with the two vaccines out on the market currently being Gardasil
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and Cervarix. The clinical trials have shown that they are one hundred percent effective

the major downside being that they can only be used before infection occurs. This

means that the mortality rate for cervical cancer is not expected to drop for another

thirty years, this twinned with the high cost of the vaccine means that researches are

continually looking at new ways to create a vaccine treating cervical cancer. (Moyle et al

2005)

12. Production of HPV vaccine

Sasagawa, et al, (1995) case study shows the production of the HPV vaccine using VLP

technology in yeast cells. The native viruses of the papuillomavirus are non enveloped

50-60nm spherical structures. This is comprised of 72 capsomers which are made up of

5 identical monomers therefore forming an iscohederal symmetrical sphere. The VLP

method aims to replicate this structure however without the viral information inside.

The genes HPV6 and HPV 16 have been analysed in this report to show a general

production method for L1 capsid proteins that form the HPV vaccine. The DNA code that

produces the L1 protein of HPV 6 are generated using the polymerase chain reaction

(PCR).This was then cloned into a sites of a pBSK vector. This same technique is also

used for the HPV16 gene which is shown in figure 2. The capsid genes of the

recombinant pBSK where then cut out and are then introduced into the yeast

expression vectors pREP which contains a thiamine repressible promoter.

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Figure 2 - HPV vector generation (Sasagawa, et al, 1995).

After an initial growth phase the monomers where expressed in vivo. To achieve

recovery the solution underwent centrifugation recovering the yeast cells. These where

then mechanically separated using glass beads in a cell breaking machine for a period of

two minutes with repeated cooling. The isolation stage occurred by using a centrifuge

again at 10,000rpm for ten minutes and then filtered for 4 minutes. The final recovery

stage uses sulfato-cellulofine chromatography. Transmission electron microscopic

analysis of the resulting mixture was then used to determine the size and production of

the VLP’s. A small sample of the mixture is placed onto a glow discharged carbon coated

grid and allowed to absorb in a moist chamber staining the cells. The mixture is then

washed out and an electron microscope is used at x34,000 magnification.

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Figure 3 - Electron microscopy of a VLP mixture without gel filtration. (Sasagawa, et al, 1995).

From figure three it can be seen that there are various VLP’s existing in the mixture with

an average size of 50nm (45-55nm) in diameter and a pentagonal capsomers structure

is shown. However, many smaller particles around 17-23nm are also seen. If a gel

filtration column is added to downstream processing after the chromatography purer

samples of the VLP are obtained as shown by figure four. This shows a much greater

uniformity throughout the VLPs with less contaminants in the fluid. Furthermore, it

underlines the importance of purification steps to increase the purity of the end product

and the effect that each stage has on the final clarity.

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Figure 4 - VLP mixture after gel filtration(Sasagawa, et al, 1995).

The L1 coding sequences can be cloned into vectors with a Ptac promoter allowing it to

be expressed in E coli. A further code(Glutathione-S-transferase GST) has to be added

onto the coding sequence to make sure that the L1 Protein would not degrade inside the

cell. Although this increased the size of the gene by 27kDA amino terminals it does not

inhibit the polypeptide ability to fold and form pentamers correctly.

13. Possible changes that can be made to the analysis methods used in VLP production
Transmission electron microscopy (TEM) allows a direct approach to be used as it

determines the characterization of the sample through visualisation therefore allowing

the user to have a more overall view of the sample. However, because of the staining

process using the glow discharged carbon coated grid and the vacuum conditions in the

microscope, distortion of the cells occur. Therefore, some VLPs can be observed to have

smaller and more distorted shapes affecting the ability to accurately measure the

sample and decide on the purity of the sample. Another disadvantage is that although

acceptable for experimental applications it requires a large amount of skill and money

to operate and is therefore very expensive for routine quality control measures. The

time required to view the samples is also very high due to the difficulty in sample

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preparation and viewing time. Field-flow fractionation (FFF) is a new idea for VLP

characterisation although it has long existed in biotechnology to characterize DNAs. It

allows a high-resolution analysis for particles ranging from several nanometres to just a

few microns.

FFF works by using a cross flow channel, which is perpendicular to the flow of the fluid.

The flow of the fluid is laminar with a parabolic velocity profile therefore having zero

flow velocity at the side of the channel wall. This cross flow channel interacts with the

particles in the channel flow causing them to flow with it. This causes a concentration

profile in the channel so that the smaller particles resist the cross flow channel and

diffuse into the areas of lower concentrations at the top of the channel, separating the

particles based on their size as shown in figure 5. This therefore causes the particles to

have different retention times in the channel.

Figure 5 - Diagram showing the FFF technique(Chuan, et al, 2008)

There are several different techniques that can be utilized by the VLP process including

thermal, electrical, sedimentation and flow FFF. However, thermal and electrical FFF

causes the sample to undergo deformation and aggregation while sedimentation FFF

has a centrifugal field causing further sample degradation. Flow FFF is therefore the

only technique which can be applied to VLP analysis as near no deformation occurs as a

result of its use. There is also minimal shear degradation of the sample as there is only a

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small amount of mechanical stress exerted on the sample in their native physical-

chemical environment. The cross flow of the fluid should be distributed along the length

of the channel uniformly as otherwise distorted flow profiles will occur decreasing the

efficiency of the separation. Asymmetrical flow field-flow fractionation (AFFFF) allows a

uniform cross flow velocity of the fluid. The porous upper wall is replaced by a solid

wall while the permeable bottom layer remains. As the upper wall is now solid the only

source of the flow is the channel flow. A cross flow therefore can be generated by use of

a pressure differential across the membrane driving the larger particles to the semi

permeable membrane which is shown in figure 6.

Figure 6 - A diagram showing the AFFF technique where A shows the focussing, injection and relaxation stages and B shows
the elution stage(Chuan, et al, 2008)
There are four steps to the process, focusing, injection, relaxation and elution. In the

focusing step the channel flow is split and is then introduced at the inlet and the outlet

of the channel and the opposing streams meet at point in the channel. A cross flow is

developed due to the pressure differential across the membrane. In the injection step

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the sample is placed in and is focussed by the funnel formed by the opposing streams.

The relaxation step is where the sample particles are separated by the cross flow

reaching equilibrium. Elution then occurs where the channel flow is only introduced at

the inlet and therefore the particles travel through to the outlet and is then measured by

multi angle light scattering (MALS). This is a non-invasive technique, which has a high

enough resolution to measure the nano particles of VLPs. This determines the molecular

mass and average size of the particle sin the solution by detecting the amount of light

scattered. This technique measures the scattered light at fixed angles. This technique

allows a large through put of liquid as the column in AFFF has a large capacity while

MALS is a very quick process and relatively inexpensive therefore minimizing the cost

to the pharmaceutical company as well as time needed. Chuan, et al, (2008), has used

various combinations of transmission electron microscopy, FFF, AFFFF and MALS to work

out which combination allows the best and quickest analysis of results. As the vaccines

produced need to undergo stringent analysis, it is important that the tools used allow small

variability’s across the batch to be detected and compared with the batches used in the clinical

trials. The method should also not alter the physical properties of the VLP samples and be

performed under a buffer condition that is native to the sample. The speed of the process is also

very important to allow accurate representation of the entire product system if the techniques

are used routinely. Chuan, et al (2008) found that the methods used in Sasagawa, et al, (1995)

report did not satisfy the above conditions. The size of the samples observed by TEM where

found to be significantly smaller than those of the samples observed by MALS. This has been

presumed to be a direct consequence of the sample preparation and viewing. This is also the

most expensive and protracted technique looked at resulting in less particles being able to be

screened and therefore worst statistics. The AFFFF-MALS technique allows sensitive,

quantitative and fast results to be processed allowing the manufacture quick updates on the

products purity.

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14. Conclusions
The human Papillomavirus is one of the most serious diseases that has so far had VLP

technology used to eradicate it. HPV causes cervical cancer which at present has the

second highest mortality rate of cancers prevalent in woman. There are two main

vaccines that can be used therapeutic and prophylactic however only prophylactic

vaccines have so far been released on to the market. The prophylactic HPV vaccine

mimics the structure of the HPV protein so therefore has 5 identical monomers making

up a capsomer, of which there are 72 in total making up the total structure. The DNA

code that makes this monomer is generated using the polymerase chain reaction and is

then cloned in the sites of pREP vector which are introduced into the yeast cells. These

are allowed to grow to a set time and then a series of isolation steps are used to purify

the VLP vaccine. An analysis step is then utilized to make sure that each batch has the

set purity needed to pass the stringent standards needed to market the vaccine.

Transmission electron microscopy is then used to evaluate the sample, however it is an

expensive and slow method of analysis and feed flow fractionation can simplify the

process and diminish the costs associated with the evaluation step. FFF allows the

material to be separated by size using a cross flow. This technique can be further

improved by using an asymmetrical flow field-flow fractionation as it allows a uniform

cross flow to occur in the cylinder. By optimizing the operating conditions any possible

aggregation of the sample can be minimized allowing the separation of molecules to

occur efficiently. Multi angle light scattering can then be used to analyse the throughput.

This technique vastly improves the speed of the process allowing greater control over

the process as the batch products can be analysed much quicker allowing changes to be

made to the production if necessary.

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15. Conclusions
This paper has explained how VLP vaccinations can be produced and by using an

example of the production of HPV in yeast cells it has helped explain how these

techniques can be improved specifically the analysis if the vaccine using FFF. VLP can

be produced using the upstream and downstream techniques shown in this report

however, the technology can continue to improve as shown by FFF. AFFF has allowed a

much faster analysis technique to be used in combination with the production of VLP

vaccines to ensure that the purity and therefore the effectiveness and safety of the virus

to measured accurately. It has been shown that VLP vaccinations are an area of

increasing interest in pharmaceutical companies with two new vaccines helping prevent

cervical cancer while many more are in the process of being developed or in their

clinical trials. VLP vaccines reduce the potential risk to the host compared to other

vaccine production methods as it does not use any viral content in the structure and

clinical trials so far have shown no major side effects to have taken place.

Although VLP based vaccines have been shown in this report to effectively work against

certain types of infections further improvements can be made to the process as well as

finding new and improved vaccination techniques. There are various new vaccinations

being considered at present including, polyvalent and therapeutic VLP vaccines.

(Stanley, et al, 2008) Polyvalent VLP vaccines are where additional genes are added to

the vaccine allowing it to have increased coverage of the virus types. Therapeutic VLP

vaccines can be added to a prophylactic vaccine preventing reactivation of the virus as

well as having an immediate impact on the viruses impact. Four such VLP-based

vaccines have been clinically tested and one has achieved proof of principle: a reduction

of blood pressure in hypertensive patients. (Jennings, Bachmann, 2008) The way the

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vaccine is delivered is also currently being considered with nano particles being used

therefore rejecting the need for needles to be used. Other types of non-invasive

techniques are also being investigated such as lower airway routes of administration.

This is seen as more effective as it is an easy and painless administration allowing mass

immunisation to happen quicker with a reduction in storage and process costs.

Although oral administration seems like an easy way to introduce the vaccine into the

host there is a high amount of VLP degradation that happens in the stomach therefore

decreasing its efficiency. As VLP technology is a very recent development VLP vaccines

still have no long term studies conducted into their safety so long term side effects can

not as yet be gauged.

For the HPV Gardasil vaccine a five year follow up study has shown a persistence of the

neutralizing antibodies however there is an approximate decline of around tenfold of

the antibodies. This shows that there need to be more in-depth studies of the vaccine

but as this decrease stabilizes after two years, the vaccine is still stable, and shows that

long lasting effects can occur. (Bharadwaj, 2009)Another disadvantage that may occur

is that it is not a therapeutic vaccine people have to be vaccinated before they may be

affected by the virus. In the case of HPV the virus transmits through sexual activity and

therefore women will have to be vaccinated mainly between the ages of 10-13 as they

are more likely to still be seronegative for HPV. However, vaccination at such an early

age requires parental approval and a recent study concluded that 23% of parents may

deny their child the vaccine. (Mays, Sturm, Zimet, 2004) This is because they believe

that as the children will have to be informed of the vaccine and therefore of the sexual

behavior that it may encourage early sexual activity. However, as the vast majority of

adult have allowed their children to have the vaccine as they see the advantages

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 Virus-like Particle Literature Review 2010
outweighing these small ethical considerations then the HPV vaccine can be seen as

successful. Furthermore these ethical considerations concern HPV and for most

vaccines parental concern will not be major problem. This report has therefore shown

that VLPs can be potentially one of the safest and fastest ways of making vaccines,

which have an enhanced immune response from humans when compared to other

vaccine techniques. There are improvements though that can be made to vaccine

process, and with future techniques being considered many more VLP based vaccines

will be created by the vaccine industry.

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 Virus-like Particle Literature Review 2010

16. References
Bharadwaj, M., Hussain, S., Nasare, V., Bhudev, C., 2009. HPV & HPV vaccination: Issues in developing
countries. Indian J Med Res, vol 130, pp 327-332

Bland. E, 2009, Virus-Like Particles May Fast-Track Vaccines, Discovery News, viewed 18 May 2009,
http://news.discovery.com/human/vaccine-virus-particles.html

Casal, J.I., 2001. Use of the baculovirus expression system for the generation of virus-like particles.
Biotechnol. Genet. Eng. Rev, vol 18, pp. 73–87.

Chackerian, B., 2007. Virus-like particles-flexible platforms for vaccine development. Vaccines. vol 6, pp
381-390.

Chuan, Y.P., 2009, Self-assembly processing of virus-like particles. PhD. Thesis, The University of
Queensland

Chuan, Y. P., Fan, Y. Y., Lua, L., Middelberg, A.P.J., 2008. Quantitative analysis of virus like particle size and
distribution by field-flow fractionation. Biotechnol. Bioeng, vol 99, pp 1425-1433.

Cruz, P.E., Maranga, L., Carrondo, M.J.T., 2000. Integrated process optimization: lessons from retrovirus
and virus-like particle production. Journal of Biotechnology, vol 88, no 3, pp188-214

Garcea, R.L., Gissmann, L., 2004. Virus-like particles as vaccines and vessels for the discovery of small
molecules. Current opinion in Biotechnology, vol 15, no 6, pp513-517

Jennings, G.T., Bachmann, M.F., 2008. Immunodrugs: Therapeutic VLP-Based Vaccines for Chronic
Diseases. Annual Review of Pharmacology and Toxicology,Vol 49, pp 303-326

Lee, C.H., Yan, Y.P., Liang, S.M., Wang, T.F., 2009. Production of FMDV virus-like particles by a SUMO
fusion protein approach in Escherichia coli. Journal of Biomedical Science, vol 16, pp16-69

Maranga, L., Cunha, A., Clemente, J., Cruz, P., Carrondo, M.J.T., 2004. Integrated process optimization:
lessons from retrovirus and virus-like particle production. Journal of Biotechnology, vol 107, no 1, pp55-
64

Mays, R.M., Sturm. L,A., Zimet, G.D., 2004. Parental perspectives on vaccinating children against sexually
transmitted infections. Soc Sci Med, vol 58, pp 1405–1413

Moyle, P.M., Barozzi, N., Wimmer, N., Olive, C., Good, M., Toth, I., 2005. Development of peptide vaccines
against HPV-16 associated cervical cancer and group A streptococci. Biopolyers, vol 80, no 4, pp556-7.

Noad, R., Roy, P., 2003. Virus-like particles as immunogens. Trends Microbiol, vol, 11, pp 438-444.

Oberholzer, M.R., Lenhoff, A.M., 1999, Protein adsorption isotherms through colloidal energetics.
Langmuir, vol 15, pp 3905-3914.

Quality and safety of vaccines, 2009, World Health Organisation,

<http://www.euro.who.int/vaccine/20081205_4>, viewed 23/05/2010

Sasagawa, T., Pushko, P., Steers, G., Gschmeissner, S.E., Hajibagheri, M.A.N., Finch, J., Crawford, L.,
Tommasino, M., 1995. Synthesis and Assembly of Virus-like Particles of Human Papillomaviruses Type 6
and Type 16 in Fission Yeast chizosaccharomyces pombe. Virology, vol 206, pp 126-135

Schiller, J.T., 1999. Papillomavirus-like particle vaccines for cervical cancer, Molecular Medicine Today,
vol 5, pp 62-68

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 Virus-like Particle Literature Review 2010
Silva, D.M., Velders, M.P., Nieland, J.D., Schiller, J.T., Nickoloff, B.J, Kast, W.M., 2001. Physical interaction of
human papillomavirus virus-like particles with immune cells. International Immunology, vol. 13, no. 5, pp
633-641

Stanley. M, Gissmann. L, Narelli-Haefliger. D, 2008. Immunobiology of Human Papillomavirus Infection

and Vaccination - Implications for Second Generation Vaccines. Vaccine, vol 26, no 10, pp 62-67

Villa, L.L., Costa, R.L.R., Petta, C.A., Andrade, R.P., Paavonen, J., Iversen, O.E., Olsson, S.E., Hoye, J., Steinwall,
M, Riis-Johannessen, G, Andersson-Ellstrom, A, Elfgren, K, Von Krogh, G, Lehtinen, M, Malm, C., Tamms, G.
M., Giacoletti, K, Lupinacci, L, Railkar, R, Taddeo, F. J, Bryan, J, Esser, M. T, Sings, H. L, Saah, A.J. & Barr, E,
2006. High sustained efficacy of a prophylactic quadrivalent human papillomavirus types 6/11/16/18 L1
virus-like particle vaccine through 5 years of follow-up. British Journal of Cancer, vol 95, pp 1459-1466.

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