Virus-like particles (VLPs) have significant applications in veterinary vaccine research. VLPs mimic the structure of viruses but lack the viral genome, making them non-infectious and safer vaccine candidates. There are three main types of VLP structures: monolayered non-enveloped VLPs composed of a single capsid protein, multilayered non-enveloped VLPs composed of multiple capsid proteins, and enveloped VLPs containing viral surface proteins embedded in a membrane. VLPs induce strong humoral and cellular immune responses through repetitive epitopes on their surface. They can also serve as carriers to present foreign epitopes and induce immunity against heterologous pathogens

1. VIRUS LIKE PARTICLES
SIGNIFICANCE IN VETERINARY RESEARCH
Eazhisai R
Post Graduate I year
Dept. of veterinary microbiology

2. DEFENITION
 Composed of one or more structural proteins
 No genomes of native viruses
 Mimic the organization and conformation of authentic virions
 No ability to self-replicate in cells
 Potentially yielding safer vaccine candidates even without the need for
any adjuvant
(Zhang et al., 2000; Georgens et al., 2005; Buonaguroet al., 2010)

3. Why VLPs?
 Well defined geometry and remarkable uniformity with repetitive surface
structures
 Particulate and multivalent nature
 Preservation of native antigenic conformation
 Safety, as they are absolutely non-infectious and non replicating
candidates
 Applicability as vectors for the presentation of foreign antigens

4. Cont.
 Higher stability than soluble antigens in extreme environmental
conditions
 Amenable to fulfil the Differentiating Infected from Vaccinated Animals
(DIVA) compliance concerns

5. PICTURE COURTESY :
E. Crisci et al. / Veterinary Immunology and Immunopathology 148 (2012) 211– 225

6. PROPERTIES OF VLPs
 Macromolecular assemblages with well defined geometry
 Usually icosahedrons or rod-like structures with diameters in the range
of 25–100 nm (Johnson and Chiu, 2000) that mimic the overall
structure of the native virions
 Composed of multiple copies of one or more viral proteins
 Antigenically indistinguishable from infectious virus or subviral
particles (Jennings and Bachmann, 2008)

7. STRUCTURAL DIVERSITY IN VLPs
 VLPs have been generated with a broad spectrum of enveloped or non-
enveloped structures, regardless of single or multiple capsid proteins
 Three major structures
- Monolayered non enveloped VLPs
- Multilayered non enveloped VLPs
- Enveloped VLPs

8. MONOLAYERED NON ENVELOPED VLPs
 These are large and constructed by expressing one major viral capsid
protein alone using a proper expression system
 Parvovirus, Picornavirus and Papillomavirus VLPs
 Monolayered non enveloped VLP based vaccine - Porcine circovirus
type 2
It consists of the ORF2 capsid proteins of native virus
and could induce broad immune protection against different genotypes and
various geographic isolates

9. MULTILAYERED NON ENVELOPED VLPs
 Reoviruses such as BTV, African horse sickness virus and rotavirus
consist of multilayered capsids without envelopes with icosahedral
symmetry
 Co-expression of multiple proteins technically difficult in host cells,
regardless of expression systems
 Belyaev and Roy, reported that co-expression of up to four major
structural proteins (VP2, VP3, VP5 and VP7) of BTV in insect cells was
successfully achieved and further led to the generation of VLPs

10. Cont.
 Limitations :
Stoichiometry and assembly efficiency of structural
proteins may be other factors influencing the formation of multilayered
non enveloped VLPs

11. ENVELOPED VLPs
 Enveloped VLPs of viruses are similarly replication-incompetent, and
the immunogen consists of assembled particles containing some or all of
the surface components of the virus embedded in plasma membrane
(Plummer and Manchester, 2010)
 McGinnes et al. (2010) constructed two VLPs for NDV vaccine :
- one containing NP, F, M and HN proteins used as an
immunogen, without adjuvant, in BALB/c mice
- other one, driven from the first one, contained the
ectodomain of Nipah virus G protein fused to the NDV HN protein
cytoplasmic and transmembrane domains

12. STRUCTURAL DIFFRENCE
PICTURE
COURTESY :
F. Liu et al. /
Comparative
Immunology,
Microbiology and
Infectious
Diseases 36
(2013) 343– 352

13. IMMUNOGENIC PROPERTIES OF VLPS
 Conventional subunit vaccines need adjuvants to elicit immune
responses, VLPs as immunogens, without any adjuvants, inducing
strong cellular and humoral responses in vivo
 A number of VLPs act as “danger signals” to trigger the innate immune
system and possess potent adjuvant activity to enhance the
immunogenicity
 They contain densely repetitive epitopes, so VLPs are commonly more
immunogenic than recombinant protein immunogens
 VLPs have an extensive potential to induce maturation of dendritic
cells and macrophages, as well as to trigger of numerous populations of
immune cells

14. HUMORAL IMMUNITY
 Repetitive epitopes of VLPs effectively be captured and processed by
APCs, subsequently cross-link the specific B-cell receptor (BCR) on the
surface of B-lymphocytes, thereby leading to B-cell activation and a
prompt T-independent IgM response
 VLPs could directly bind and interact with naive B cells in vitro, thus
probably causing the expression of activation markers CD69 and
CD86, plasma cell formation, specific antibody production and
IgG2a class switching both in vitro and in vivo

15. CELL MEDIATED IMMUNITY
Induction of cytotoxic T lymphocyte (CTL) responses by dendritic cells
in the absence of viral replication
Priming of CD8+ T cells
Class I MHC
Exogenous VLPs

16. Cont.
 VLP directly induce the phenotypic and functional maturation of
dendritic cells, causing the upregulation of co-stimulatory molecules
and cytokines, which enhance activation of CD8+ T cells
 Lechmann et al. showed that immunized BALB/c mice with hepatitis
C VLPs developed virus-specific cellular immune responses including
CTL and T helper responses with gamma interferon production

17. EE - early Endosome
LE - late endosome
LS - lysosome
MHC - Major
Histocompatibility
Complex
RER - rough
endoplasmic
reticulum
TV - transport vesicle
VLP – virus like
particle

18. GENERATION OF ANIMAL VIRUS VLPs
 VLPs are generated through the co-expression and then self assembly of
their components in yeasts (Freivalds et al., 2011), Escherichia coli
(Yin et al., 2010), mammalian cells (Wuet al., 2010) and insect cells
(Baek et al., 2011)
 Insect cells, baculovirus based expression system(BES) plays a key
role in self assembly and release of VLPs (Ye et al., 2006; Luo et al.,
2007; Pillay et al., 2009; McClenahanet al., 2010).Because of the high
expression levels of insect cell expression systems in comparison to
mammalian cell expression systems, and the versatility of the BES for
expressing VLPs formed by multiple proteins

19. (a) Both linearized AcNPV baculovirus DNA and
recombinant transfer vector are co-transfected
into Sf cell and recombination occurs
(Sf: Spodoptera frugiperda)
(b) Recombinant baculovirus
(c) Recombinant viruses are harvested and
amplified to infect insect cells
(d) The foreign genes express the proteins of
interest, respectively
(e) The proteins of interest self-assemble into
VLPs by interaction with each other within the
Cytoplasm
(I) recombinant transfer vector
(II) linearized AcNPV baculovirus DNA
(III) recombinant baculovirus DNA
(IV) recombinant baculovirus
(V) proteins of interest.
PICTURE COURTESY : F. Liu et al. / Research in Veterinary Science 93 (2012) 553–559

20. VLPs as CARRIERS OF HETEROLOGOUS
EPITOPES
 VLPs can be used to induce immune responses against heterologous
antigens
 Due to their particulate nature, it can be employed to deliver additional
antigenic structures, such as whole proteins or specific individual
epitope, to induce more effective immune responses than their soluble
counterparts (Buonaguro et al., 2006)
 Their capsids serve both as a presentation scaffold for epitopes from
another viral, bacterial, or parasitic pathogen, and as an adjuvant to
boost the immune response(Plummer and Manchester, 2010)

21. CHIMERIC VLPs
 The insertion of target epitopes into viral structural proteins to generate
chimeric particles, named as chimeric VLPs, which displaying
heterologous epitopes on VLPs
 Advantage of the chimeric VLPs is that the target epitopes may be
displayed in the same conformation and at high density on the particle
surface through successful incorporation

22. Cont.
 Limitations :
The production process of chimeric VLPs is highly unpredictable,
depends many factors, including the type of chemical bonds established
between proteins, the glycosylation efficiency and cell type
(Roldao et al., 2010)
 Importantly, in order to elicit sustained high-titer antibody responses
against heterologous pathogens, foreign proteins should be compatible
with structural proteins of chimeric VLPs

23. DIFFERENTIATING
INFECTED FROM VACCINATED ANIMALS (DIVA)
 Conventional vaccines, such as live attenuated vaccines and inactivated
vaccines, may not be used to differentiate infected from vaccinated
animals, since antibody responses induced by such vaccines are
generally same as those induced by wild type viruses
 DIVA (differentiating infected from vaccinated animals) vaccine,
originally known as marker vaccine, usually based on the absence of at
least one immunogenic protein in the vaccine strain, allows DIVA in
conjunction with a diagnostic test that detects antibodies against the
antigens lacking in the vaccine strain

24. Cont.
 The first licensed genetically engineered DIVA vaccine was introduced
in 1988 with a companion glycoprotein E blocking enzyme-linked
immunosorbent assay (ELISA) kit to detect wild-type pseudorabies virus

25. ROLE OF VLPs IN DIVA
 VLP based vaccine candidates offer a promising strategy for DIVA, as
VLPs lacking either monovalent or multivalent antigens can be
constructed on the need for serological surveillance
 A VLP, if devoid of at least one immunogenic protein present in the
corresponding field virus, is consequently sometimes referred to as a
negative DIVA vaccine
 A VLP, containing an additional immunogenic substance compared with
the corresponding field virus, can be defined as the positive DIVA
vaccine

26. AIV VLPs composed of the HA and
M1 are produced using baculovirus
expression system in Sf9 cells. The
antibody (A) against the HA is
induced by vaccination with VLPs;
the antibodies (A and B) against
both the HA and NA are induced by
natural infection with field AIVs.
Using a companion ELISA
detecting the antibody against NA,
it is possible to distinguish infected
from vaccinated chickens.
A - antibody against hemagglutinin
AIV - avian influenza virus
B - antibody against neuraminidase
BV - baculovirus
BVG - baculoviral genome
PICTURE COURTESY : F. Liu et al. / Comparative Immunology,
Microbiology and Infectious Diseases 36 (2013) 343– 352

27. Cont.
 Limitations :
- ELISA tests, are exclusively based on the detection of antibodies
induced by the wild type pathogen
- Detection of differentiating antibodies in animals is only possible
weeks after the acute virus infection
- Many antigens designed for the purpose of DIVA vaccination may
be weakly immunogenic
- Titer of DIVA specific antibodies induced by such antigens
keep a relatively low level that cannot be detected with conventional
diagnostic tests

28. CHALLENGES FOR VLP BASED VACCINE
DEVELOPMENT
 Manufacturing process is not scalable or cost-effective
(Buckland, 2005)
 VLPs made by single protein assembly are able to be produced in
large amounts and high quality, whereas structurally complex VLPs
are raise difficulties for large scale production
(Cox, 2012; Mena and Kamen, 2011;Roldao et al., 2011)
 Inherent properties of the lipid envelope, made the production of
enveloped VLPs is technically more complex
(Roldao et al., 2011)

29. Cont.
 Baculovirus Expression System(BES) - most popular
- Drawback of BES is the significant coproduction of
infective baculovirus particles, which are difficult to separate from VLPs.
The baculovirus particles can interfere with the immunogenicity of the
VLP-based vaccines (Hervas-Stubbs et al., 2007)
- VLP based immunogens produced in the baculovirus
expression system must undergo either chemical inactivation treatments
to eliminate baculovirus infectivity that may impair the quality of the
produced VLPs (Rueda et al., 2000)

30. Cont.
 VLPs foreign epitopes displays only epitopes of a limited size to be
targeted. Since pathogens usually undergo antigenic variation in
response to host immune pressures, vaccines based on VLPs only be
effective against highly conserved B or T cell epitopes

31. VLP BASED VACCINE
 Porcine circovirus type 2 (PCV2) VLP-based vaccine Porcilis PCV®
(manufactured by Intervet International, The Netherlands), is licensed
and commercially available (Mena and Kamen, 2011)

32. THANK YOU