Download PEP_2011_13_Recombinant vaccine

Recombinant vaccines
Dr.Alain Jacquet (Chulalongkorn University)
Vaccines
Represent one of the greatest triumphs of modern medicine
Vast majority of vaccines have been developed empirically, with little or no
understanding of the immunological mechanisms by which they induce
protective immunity
The need to understand the immunological mechanisms by which vaccines
confer protective immunity ( failure to develop vaccines against HIV, TB,
malaria)
Two groups of conventional vaccines: live attenuated and killed vaccines
Most vaccines are believed to confer protection through neutralizing
antibodies (memory B cells)
But critical role of T-cell responses (memory T cells) for protection also
The best: Antibody-based and T-cell-based vaccines!!
Successful vaccines: based on antibodies against pathogens with
stable antigen repertoire
To date, fail to develop vaccines against HIV, malaria and tuberculosis because
of antigenic variability and the requirement of T-cell immunity for protection.
Success rate on vaccine development
MMR, measles, mumps and
rubella; HBV, hepatitis B virus; HAV,
hepatitis A virus; HIB, H. influenzae
type B.
IPV, inactivated poliovirus; OPV, oral poliovirus;
Live attenuated Vaccines
Attenuated strains almost or completely devoid of pathogenicity but able of
inducing a protective immune response. Replicative in the human host and
provide continuous antigenic stimulation over a period of time,
Attenuation methods for vaccine production.
Use of a related virus from another animal ( ex: cowpox to prevent
smallpox)..
Administration of pathogenic or partially attenuated virus by an unnatural
route (ex: vaccine against adenovirus using enteric route).
Repeated passages of the virus in an "unnatural host" or host cell (main
methods for the major vaccines used in man and animals). Ex: yellow fever
17D strain developed by passage in mice and then in chick embryos.
Polioviruses passaged in monkey kidney cells and measles in chick embryo
fibroblasts
Inactivated/killed vaccines
Easiest method for vaccine preparation
Inactivation with heat and/ or chemicals
(usually formalin).
Non-living, non-replicative
Need much more antigen than live
vaccines able to replicate in the host
Kinetics of poliovirus inactivation with formaldehyde. Determination of virus titre by the plaque
assay method (A) and detection of poliovirus RNA by RT-PCR (B)
Comparison Live-attenuated vs killed vaccine
Live-attenuated
High
Killed
Weak
Dose
Number of dose
Low
Single
High
Need for adjuvant
Duration of immunity
No
Many years
CMI
Good
Risky
Poor
Not possible
High
Low
Global immune
response following 1
injection
Reversion to
virulence
Cost of production
Multiple
(prime/boost)
Usually
Less (recall
injection)
Safety and production problems
Live-attenuated
Under-attenuation
Killed
Incomplete inactivation
Genetic mutation leading to
reversion to virulence
Increased risk of allergic
reactions due to large amounts
of antigen involved
Preparation instability
Failure to grow large amounts of
organisms in laboratory
Contaminating viruses in
cultured cells
Excessive treatment can
destroy immunogenicity
Heat lability
Immune response weaker
Not for immunocompromized or
pregnant patients
increasing the expense and
reducing vaccine coverage
The Pasteur’s methods and new technologies
From Live attenuated/killed vaccines to Virus-like particles (VLPs),
Engineered replicating and nonreplicating recombinant vectors
and Subunit vaccines.
Recombinant subunit vaccines
Defined recombinant antigens (HBsAg, L1-HPV-16) which are the best to
stimulate immunity
Advantages
Production and quality control simpler
Not associated with other microbial proteins or DNA
No potential for replication
Safer in cases where viruses are oncogenic or establish a persistent infection
Feasible even if virus cannot be cultivated
Disadvantages:
May be less immunogenic than conventional inactivated whole-virus vaccines
Requires adjuvant
Requires primary course of injections followed by boosters
But also toxoid vaccines (as inactivated diphtheria and tetanus toxin),
carbohydrate vaccines (pneumococcus), conjugate vaccines (Protein carrier +
carbohydrate as H. influenzae type B or meningococcus).
Expression systems for recombinant subunit vaccines
Many antigens are glycosylated: E.coli usually not efficient
Glycosylation can influence immunogenicity
Subunit vaccines are combined with adjuvants to improve
immunogenicity.
Effect of major adjuvants on the humoral and cellular responses
Adjuvants act through recognition by pattern-recognition receptors
(PRRs) to activate INNATE IMMUNITY
C-type lectin
receptors
triggering receptors
expressed on
myeloid cells
Nucleotide-binding and oligomerization
domain (NOD)-like receptors
Retinoic acid-inducible
(RIG)-like helicases
Virus-like particles (VLP)
Natural ability of many types of viral capsid subunits to self-assemble into
virus-like particles (VLPs)
Consist of a noninfectious subset of viral components, but mimic killed
vaccines since they typically present a whole, but inactive virus particle to the
host
Safer than killed or attenuated virus vaccines.
(a) Icosahedral capsid of human papilloma
virus (HPV)16 L1 capsid
(b) Electron micrograph of HPV virus-like
particles (VLPs)
(c) L1 pentamer of HPV
(d) Human hepatitis B viral capsid (HBcAg)
VLPs immunogenicity
High VLPs immunogenicity
Particulate structures much more stable than soluble antigens
Repetitive surface antigen (as wt virus), a potent stimulator of B cell
responses
Induction of innate immunity through PRRs activation (VLPs structure not
typical of host proteins, putative nucleic acid encapsidation-TLR ligands)
Uptake of VLPs by APCs more efficient than soluble antigens (antigen
particules vs monomers) → stimulate MHC Class I and Class II responses →
prime lasting T-cell and antibody responses
VLPs and heterologous epitope display
VLPs as a platform for multivalent heterologous epitope display to mount an
immune response to the protein or peptide attached through fusion or through
genetic insertion into the capsid
presentation scaffold for epitopes from another viral, bacterial, or parasitic
pathogen, and as an adjuvant to boost the immune response
Issues about the large epitope size that might hinder VLP assembly
FHV (Flock House virus) chimera 264
capsid protein (green) and anthrax
binding domain (yellow, left) and model
including the protective antigen fragment
bound to the surface (purple, right).
Bacteriophage Qβ capsidbased VLP for antigen
conjugation
New recombinant vaccines by gene deleterious mutation
Advances in molecular virology→ identification of many viral genes
associated with virulence and immunogenicity
Deletion or mutation of these genes results in a ‘defective virus,’ which cannot
replicate in the host or spread to better control the replication and pathogenesis
of vaccine candidates
Novel attenuated virus very limited viral replication and antigen production
Host immune response not required to limit viral spread → New recombinant
virus safer than classic LAVs, even in immunocompromised patients.
Replication-defective
virus
One or several genes
required for genome
replication deleted in the
vaccine strain. Virus
vaccine produced in helper
cell line that express the
missing protein(s) in trans
Administrated virus unable
to replicate
Single-cycle virus
One or several genes
required for viral assembly
and spread deleted in the
vaccine strain. Still
competent for genome
replication
Virus able to replicate its
genome but defective for
assembly or spread
Defective viruses propagated in
‘helper’ cells that express the missing
gene(s). In the inoculated host (normal
cell), the virus is unable to replicate its
genome but viral genes are still
expressed, which can induce a strong
immune response
‘Single-cycle viruses are defective in a
viral protein required for assembly or
spread. Although these viruses can
replicate their genome through a single
cycle, no production of infectious virus.
Issue at the level of the vaccine
efficiency for human disease.
Quite weak immune
response
because the antigen is only expressed
at the site of inoculation.
Safety
concerns
about
the
completeness of the block in viral
spread in single-cycle viruses.
Difficulty to balance immunogenicity
with safety,
Attenuation by codon deoptimization
Many organisms exhibit a codon bias, using some codons more frequently
than others for gene expression
In bacteria and simple eukaryotes, codon preference related to amounts of
the corresponding transfer RNA and affects translational efficiency, less clear in
mammals.
Codon described in many viral genomes. Most mammalian viruses:
preference for CpG dinucleotides (with overall GC content highly variable
however)
Goal: to replace up to 50% of the capsid codons with synonymous codons
that are less preferred in the human genome.
Codon-deoptimized polioviruses (by gene synthesis) attenuated by 1,000fold on a per-particle basis compared with wild type. Because all changes are
synonymous, the proteins expressed from codon-deoptimized viruses are
identical to wild type and similarly immunogenic
Advantages.
Attenuation not affecting antigenicity (mimic natural infection).
Easily applied to any viruses.
Codon-deoptimization→ hundreds of point mutations→ little risk of reversion
to virulence
Genetically stable and remain attenuated after repeated passage.
Codon use statistics in synthetic
poliovirus P1 capsid design
PV-AB capsids, the use of nonpreferred codons
was maximized
miRNA-controlled vaccine strategy
Viral replication regulated in a tissue-specific manner by incorporating miRNA
target sites into the viral genome. In cells that express the miRNA (e.g., brain,
top cell), the miRNAs are processed and transported to the cytoplasm to
mediate cleavage of viral RNA. Viral replication restricted to cells not
expressing the miRNA.
Engineered virus trigger a natural immune response in target tissues without
the associated risk of dissemination and disease.
Ex: Poliovirus replicates in many
tissues
But disease linked to lytic infection of
the central nervous system (CNS).
Incorporation of binding sites for
miR124 (a CNS-restricted miRNA) into
the RNA genome of poliovirus
No viral replication in the CNS (murine
model of infection) MiR124-targeted
poliovirus able to replicate in nonneuronal tissues and stimulate a strong
neutralizing antibody response after a
single intraperitoneal inoculation.
Protection from subsequent challenge
with 10,000 times the lethal dose of
wild-type virus.
Recombinant viral vector for vaccine production
To convert a virus into a recombinant vector (replication defective)
Viral genome containing genes for replication, production of the virion, and pathogenicity
of the virus flanked by cis-acting sequences (providing viral origin of replication and the
signal for encapsidation)
The packaging construct contains only genes that encode functions required for
replication and structural proteins.
Vector construct containing cis-acting sequences and the heterologous antigen cassette
Co-transfection packaging + rec vector into the packaging cell by transfection, Or
generation of stable packaging cell lines. Proteins required for replication
and assembly of the virion expressed from the packaging construct, encapsidation of
recombinant genome into virus particles to generate the recombinant viral vector.
Advantages of viral vectors
Induction of antibody responses but also systemic T-cell responses
(polyfunctional cytokine-secreting CD4+ and CD8+ T-cells). Excellent for the
control of intracellular pathogens and cancer (not strongly induced by proteinbased subunit vaccines).
T-cell inducing vaccines directed to highly conserved epitopes→ potentially
offering protection against several strains of the same pathogen.
Issues:
Safety, particularly for replication competent viral vector. Often highly
immunogenic as wild-type, but also carry the risk of recombination,
reactogenicity or reversion to virulence.
Production at large scale and stability
Pre-existing immune response against the viral vector
Adaptive immune responses to the vector also blocking or reducing the
induction of the desired responses against the vaccine antigen.
→knocking out viral genes evolved to reduce host antiviral immune
responses to reduce immune evasion properties and improve adjuvant-specific
characteristics of viral vectors
Immune responses induced by recombinant viral vector
or DNA vaccine
Typical vaccine viral vectors
MVA: Modified Vaccinia Ankara, does not replicate in mammalian cells.
(MVA passaged more than 570 times in chick embryo fibroblasts, lost about
15% of its genome)
Imojev (recombinant vectored-vaccine against Japanese
Encephalitis-Sanofi Pasteur ): first licensed viral vector-based
vaccine for use in humans (Australia, end 2010)
of Japanese encephalitis virus
Attenuated 17D yellow fever virus as a vector. YFV-17D genes coding for main
surface antigens (PrM and E) replaced by the corresponding genes of JE
Viral vector veterinary vaccines in the market (2010)
Attenuated poxviruses:
ALVAC:attenuated canarypox virus
FPV: fowlpox virus
NDV: Newcastle Disease Virus
HVT: Turkey herpesvirus
IBDV: infectious bursal disease virus
Heterologous Prime-boost immunization
To increase the potency of vectored vaccines
Prime(s) given with one type of vaccine (usually DNA vaccine) and boost(s)
with another type of vaccine (viral vector or recombinant protein)
Generally: DNA vaccines alone not immunogenic enough to generate sufficient
T cell responses to protect against difficult diseases in humans
Vector-based vaccine: host responses to the structural proteins of the viral
vector itself→ reduced responses against the vaccine insert during multiple
immunizations.
Prime–boost protocols to overcome these 2 issues.
Ex : DNA–MVA, DNA–NYVAC, FPV–MVA, influenza–MVA, AdV–MVA,
heterologous AdV–AdV, and DNA–Sendai virus for malaria, HIV-1, tuberculosis
(TB) and hepatitis C through to cancer vaccines
Immunological explanation for the increased immune responses in prime boost
protocols not well understood.
Lack of additional viral antigens (of the vector) during DNA → to focus the
immune response on the key antigen.
Additional innate antiviral responses, and cytokine milieu following the viral
vector boost, may augment the booster response.
Recombinant protein as a boost to increase the antibody responses (DNA
vaccines better for CTLs than antibodies in higher species).
Viral-vector vaccines and prime–boost immunization in clinical trials
The first viral-vectored vaccine against HIV to show efficacy in humans!
Thai efficacy trial of a prime-boost regimen: ALVAC-HIV (gp120, Gag, Protease
– Sanofi Pasteur) followed by a gp120 subunit in Alum (AIDSVAX B/E, Global
Solutions for Infectious Diseases)
Statistically significant trend towards preventing HIV infection in an at-risk
population.
AIDSVAX B/E vaccine achieved protective immunity as a booster, despite the
previous lack of efficacy of AIDSVAX B/E alone in a Phase III trial.
However: Vaccination did not
affect the degree of viremia or
the CD4+ T-cell count in
subjects in whom HIV-1 infection
was subsequently diagnosed.
Vaccination with ALVAC and
AIDSVAX to prevent HIV-1
infection in Thailand. N Engl J
Med 2009, 361:2209-2220.
Do not forget the importance of the route of immunization!
Reverse vaccinology:a genomics-enabled approach to
vaccine development
Complete genome of bacteria/viruses/parasites: large reservoir of genes
encoding for potential antigens that can be selected, screened and tested as
vaccine candidates.
Potentially surface-exposed proteins can be identified in a reverse manner,
starting from the genome rather than from the microorganism = “reverse
vaccinology”
tested by FACS and ELISA to
evaluate the surface localization of
the antigens
Reverse vaccinology vs typical vaccine development
Vaccine development against Serogroup B Neisseria meningitidis
(MenB)
HTP analyses applied to vaccine development
Genome/ pan-genome (complete gene content of
organism/species, including the complete repertoire
of antigens capable to be expressed.
Transcriptome: complete set of
RNA transcripts expressed by an
organism under a specified
condition
Proteome: complete set of
proteins expressed by an
organism under a specified
condition
Surface proteome: subset of
proteins that are surface
exposed
Structural genome: 3D
structure of the proteins of an
organism, in particular the
structural epitopes of
immunogenic antigens
Immunoproteome: set of
antigens that interact with the
host immune system
Vaccinomics: Response of
individual host immune systems
respond to a vaccine)
Proteomic strategy to identify surface-exposed proteins
as new mouse-protective antigen
Gene cloning
RecProtein
production and
purification
Proteomic studies of bacterial pathogens