Virus Like

Particles
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 Contents
Introduction

Brief history

Methods to study VLPs

Examples

Applications

Conclusion

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Introduction
 Viruses are:

 “ acute infectious agents, obligate intracellular parasites” .

 Minimal structural features of a typical virus consist of one molecule of

nucleic acid encased in a protective capsid that is assembled from several
identical proteins.
 Complex viruses have genomes made up of several nucleic acid molecules.

 Their capsids may be formed by several different structural proteins, which

can be surrounded by a lipid membrane envelope acquired from the cell
membranes, or functional proteins involved in virus replication.

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Cont……
Poxviridae and Retroviridae family;
are complex viruses whose structures are not fully
understood.
Simple viruses have either of the following symmetries;
helical or icosahedral.

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Brief history
The study of virus assembly dates back to 1955.
 Fraenckel-Conrat and Williams

 Demonstrated that the simple capsid of the rod-shaped TMV could

be reconstituted in vitro from separated RNA and protein
components.
 In fact, 2130 copies of the 158 amino acid coat protein (CP)
self-assemble in a helical viral particle (300nm in length).

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Virus like particles
 The assembly of the viral structural proteins into organized
macromolecular structures (capsids) generates viral “empty
shells” known as virus-like particles (VLPs).
 Lack viral nucleic acid and are noninfectious.Virus-like
particles (VLPs) are inert, empty viral capsids, which contain
no DNA/RNA from the virus itself.
 However, they retain the structure of a virus particle and can be
engineered to have antigens attached.
 By extension, particles that contain antigens from viral or non-
viral sources and show similar size and shape as viruses are also
regarded as VLPs.
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Methods to study VLPs
 Remarkable progress in different areas of structural virology and
in understanding the assembly, shape,and functional properties
of different VLPs has been made possible by following methods:

 Cryo-electron microscopy (cryoEM)

 X-ray crystallography
 Biochemical methods like:
 Mutational analysis of the structural subunits

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Examples
 VLPs derived from the Hepatitis B virus and composed
of the small HBV derived surface antigen (HBsAg)
were described over 40 years ago from patient sera.
 VLPs from other virus families include;
 Parvoviridae (e.g. adeno-associated virus)
 Retroviridae (e.g. HIV)
 Flaviviridae (e.g. Hepatitis C virus)
 VLPs can be produced in;
 Mammalian cell lines
 Insect cell lines
 Yeast, and
 Plant cells
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Applications of VLPs
As vaccines

As vessels and display systems

Heterologous epitope presentation

VLPs as immunogens

VLPs and nanoparticle biotechnology

VLPs and Dengue virus prevention

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VLPs as vaccines
 Before 1969 anti-viral vaccines used were;
 inactivated viruses (e.g. rabies vaccine)
 attenuated strains of pathogenic wild-type viruses (yellow fever or polio) or
 were using closely related but non-pathogenic virus strains to induce a protective
immune response against the pathogenic relative—like smallpox vaccine containing
the innocuous vaccinia virus
 Discovery of HBsAg in hepatitis B patients resulted in production of potent vaccine
against cognate infection
 VLPs because of their particulate nature have advantage over soluble antigens that
have been failed to be used as vaccines because of weak immunogenicity or
instability
 The fact that VLPs can induce protective antiviral immune responses without having
the risk of infection represents a major advantage for the design of safer vaccines.
 Do not replicate and are not infectious so represent a safer alternative to attenuated
viruses.
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Cont..
 VLPs from rotavirus and Norwalk virus (NV) have
been extensively studied and cause acute
gastroenteritis in man and animals.

 Can generate a strong general humoral and mucosal

immune response.

 Effective vaccines against hepatitis B virus (HBV),

the human papillomavirus (HPV), and NV.

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Examples of various virus capsids that have been developed as virus-like particle (VLP) vaccines and
platforms for heterologous antigen. PDB IDS: hepatitis B capsid, 2QIJ; Papillomavirus (PV), 3IYJ;14,15
Hepatitis E virus (HEV), 2ZTN; Cowpea mosaic virus (CPMV), 1NY7; Alfalfa mosaic virus (AlMV), AMV;
bacteriophage Qb capsid, 1QBE; bacteriophage MS2–2MS2; Flock house virus (FHV) VLP, 2Q26.
(Tobacco mosaic virus (TMV) image has been reprinted with permission from Ref 16. Copyright © 2007
Academic Press). (Other images have been reprinted with permission from Ref 17. Copyright © 2009
Oxford University Press).

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HBsAg
The HBV surface antigen (HBsAg) can self-
assemble into 22 nm lipoprotein VLPs containing
about 100 HBsAg-S molecules without the
contribution of nucleocapsid.
Saccharomyces cerevisiae
First example of a vaccine created in a
recombinant system that is effective against a
human viral infection.
Currently in the US, Merck & Co. and
GlaxoSmithKline (GSK) are marketing FDA
approved yeast-derived HBV-like particles, known
commercially as Recombivax HB and Energix-B.
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 HP (Human Papillomavirus)
VLPs
 “non-enveloped, icosahedral, 55nm in diameter,
consisting of a regular array of 72 pentameric
oligomers composed of the major coat protein, L1.”
 Expression system: Saccharomyces cerevisiae
 Able to induce a strong B-cell response at a very low
dose even without the use of adjuvant
 The Merck vaccine, called Gardasil, targets HPV types
16 and 18, which cause about 70% of cervical cancer
cases,
 Types 6 and 11 which cause about 90% of all cases of
genital warts
 The GSK vaccine called Cervarix is a bivalent vaccine
targeting HPV 16 and 18, produced by the baculovirus
system in insect cells. 6/15/2022 14
Cont.

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NV like particles
 Norwalk virus (NV) belong to Caliciviridae family
 Causes epidemic acute gastroenteritis in humans
 Vaccine:
 Efforts to develop vaccines against NV infection led to
the discovery of Norwalk virus capsid protein (NVCP)
expressed in insect cells yield VLPs
 Made from a single recombinant coat protein
 Composed of 90 dimers of the virus coat protein
 Expression system: Baculovirus
 23nm, react with sera from infected humans and
stimulate serum responses in humans.

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 Advantages:
 Are a very promising orally delivered vaccine
 Stable:
At low pH and
To lyophilization
 Easily produced and purified in large quantities

 VLP vaccine stimulates:

In mice In humans

Humoral,
Systemic and
mucosal, and
mucosal
cellular
immune
immune
response
response
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 2. VLPs as vessels and
display systems
 Can be used as:
 Vessels for the delivery of small therapeutics
 Display systems for biologically active molecules
 Attractive use in transporting viral vectors for:
 Gene therapy and DNA vaccines exploiting in some
cases the natural tropism of the viral particles

 Super coiled plasmid DNA up to 17 kb was

encapsidated in vitro inside simian virus 40 capsid
proteins
 HPV-16 L1 VLPs are able to package unrelated
plasmid DNA in vitro and then to deliver this foreign
DNA to eukaryotic cells.
 The gene transfer rate observed was higher
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 3. Heterologous epitope
presentation
 Another attractive use of VLPs

 Chimeric HBsAg particles displaying a poliovirus

neutralization epitope stimulated the production of
neutralizing antibodies against poliovirus.

 Another fusion of selected epitopes from HIV-1 and HCV

to the HBsAg were immunogenic in rodents and primates.

 A fusion between a 9 amino acid peptide derived from the

Japanese encephalitis virus (JEV) E protein and a C-
terminal truncated version of the Johnson grass mosaic
virus (JGMV) generated chimeric VLPs in Escherichia coli.
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4. Immunogenic properties of
VLPs
 Compared with soluble antigens, which need to be co-
administered with adjuvant in several booster injections to
elicit protective immune responses
 VLPs are capable of inducing strong cellular and humoral
responses as direct immunogens.

 VLP size appears to be favourable for uptake by dendritic

cells (DC) via macropinocytosis and endocytosis that play a
central role in activating innate and adaptive immune
responses.

 Gag VLPs containing many immunogenic epitopes are

capable of stimulating cellular immune responses via both,
the MHC class-I and MHC class-II pathway

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 Model for the activation of dendritic cells
(DCs) by baculo-derived VLP preparations 21
(B) VLP-mediated
maturation of DCs.
 Uptake of VLP/BV
 Activates DC via danger
signals
 Resulting in upregulation
of DC maturation
markers.
 Mature DCs present VLP-
derived antigens to naive
CD4+ and CD8+ T cells
via MHC class-I and
class-II.
 Secretion of cytokines by
DCs stimulates
differentiation into B and
T effector cells resulting
in antibody release and
cytotoxic T cell (CTL)
responses.
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 5. VLPs as scaffolds in
Nanoparticle
biotechnology
 Nanoparticles basically encompass all particulate
structures ranging between 5 and 100 nm in size.

 Especially the capability of virus-derived nanostructures to

assemble into highly organized regular arrays together
with their susceptibility to accept a wide range of chemical
modifications has offered new perspectives for the usage of
VLPs in manifold biotechnological processes.

 Have formerly exploited the possibility to chemically

modify mutant cowpea mosaic virus (CPMV) particles–
which are highly stable and offer multiple reactive sites –
by exposing sulfhydryl groups in order to attach
fluorescent dyes and gold clusters.
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Cont.
 Likewise, the tobacco mosaic virus (TMV)-derived capsid
protein has proven to be a suitable scaffold for extensive
chemical modification allowing assembly of
nanobiopolymers

 Yet another study has described the usage of recombinant

M13 phage particles as organic templates to polymerize
nanowires as building blocks for semiconductors or
magnetic materials

 In all these approaches, chemically modified VLPs

maintained their structural integrity that is a prerequisite
for VLPs to prevail in the future world of nanoscience
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 6. VLPs and DV prevention
 Experiments were carried with mice by using VLPs
derived from different recombinant dengue-2 and
dengue-4 capsid and envelop proteins

 Conducted to check the induction of both humoral and

cellular immune responses

 Results demonstrated the suitability of VLPs to induce

a proper immune response against dengue virus

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 7. VLPs and HCV
 Development of a vaccine against hepatitis C virus via VLPs

 CIGB-230 is a vaccine candidate based on the mixture of:

 recombinant truncated HCV core protein
 able to self-assemble into VLPs
 mainly elicits antigen-specific CD4+ T cell response

 Plasmid for DNA immunization, pIDKE2, that expresses the HCV

structural antigens (Core, E1, E2)
 Induces both humoral and cellular immune responses against
Core, E1 and E2

 Enhanced functional immune response observed after

administration of the vaccine candidate CIGB-230 may be due to a
synergistic action between its two components, plasmid and
protein, perhaps due to the reduced degradation of plasmid DNA
and to a better activation and antigen presentation
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presenting cells
Conclusion
 On the basis of their flexibility and stability, simple
production and distinctive immunogenic properties VLPs
offer vast opportunities of application in the fields of vaccine
development, gene therapy as well as nanobiotechnology.

 Many lessons have been learned from VLP-based

technologies, which will certainly find themselves confronted
by new challenges in the near future, such as the demand
for innovative biomaterials or potent vaccines for newly
emerging diseases.

 In this light it appears almost ironical that viruses as such

may serve a good purpose in the biotechnological era
exploiting their weapons to beat them at their own game.
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