Principles of antiviral vaccine design

Dr Ewelina Król
Vaccination is one of the most important elements in combating many infectious diseases
caused by viruses
 VACCINES:
Vaccines are divided due to:
Type of vaccine antigen:
- live attenuated vaccines (contain live, weakened microorganisms),
- inactivated vaccines (contain killed microorganisms),
- subunit vaccines (contain broken microorganisms or their fragments);
- recombinant vaccines (contain antigens obtained by genetic engineering),
Content:
- monovalent vaccine (contain one antigen),
- Polyvalent vaccine (contain several antigens),
- combined vaccines (contain toxins, killed bacterial cells, viral antigens and
viruses);
Types of vaccines
Live attenuated vaccines (weakened strains of primary pathogenic
microorganisms with minimized virulence):
- using a related virus from another animal (use of cryopox virus)
- multiple passage of the virus in the "unnatural host" or in host cells under
conditions favor to mutations and the emergence of strains with new
properties (yellow fever strain 17D passaged in mice and then in chicken
embryos; polio viruses were passaged in monkey kidney cells and
measles virus in chicken embryo fibroblasts)

Inactivated vaccines:

- Inactivation by high temperatures or chemical agents (formaldehyde or

beta-propiolactone).
Efficacy vs safety:
Sridhar et al., 2015, Vaccines 3, 375-389
Recombinant vaccines antigens
• Split virus antigens
• Recombinant proteins
• VLPs/nanoparticles
• Toxoids
• Conjugates
• Outer Membrane Vesicles (OMVs)
• Others…
Characteristics of a good vaccine:
• security,
• effectiveness (long-term protection),
• simple application,
• no side effects,
• durability=stability,
• low cost,
• High production efficiency.
Antiviral vaccines available on the market:

Live attenuated Inactivated Recombinant

measles, rubella, tick-borne encephalitis, hepatitis B, influenza,

mumps, chickenpox, hepatitis A and B, human papilloma virus,
shingles, yellow fever, rabies, polio, influenza SARS-CoV-2
polio, rotavirus,
influenza
 Top 5 best selling vaccines in the world:

Pneumococci

HPV

Diphtheria, tetanus, pertussis,

polio, haemophilus influenzae
type B (Hib)

Influenza

Meningococci
mRNA vaccines
 Production of mRNA vaccines

DNA
plasmid

RNA
polymerase

in vitro transcription Lipid nanoparticles

https://pl.promega.com/resources/pubhub/2019/tpub-211- Kauffman K.J. i wsp., Nano Lett. 2015; 15(11):7300-6

the-many-uses-for-in-vitro-transcribed-rna/
RNA modyfications in mRNA-1273 (Moderna) and
COMIRNATY (Pfizer/ BioNTech) vaccines

• 2P mutations – preservation of pre- Pre-fusion Post-fusion

fusion protein S structure structure structure

• Use of modified nucleotides

Pardi, N. i wsp. 2018, Nat Rev Drug Discov 17: 261–279 https://cen.acs.org/pharmaceuticals/vaccines/tiny-tweak-behind-COVID-19/98/i38
Mechanism of action of mRNA vaccines

T cell

RNA in lipid
nanoparticles

muscle cell

T cell CD8+

T cell CD4+ B cell

antibodies

Chmielewska A., Mrukowicz J. 2021, Medycyna Praktyczna 1/2021

Replicating vectors
 Replicating vectors
ADVANTAGES:
- High transduction efficiency
- High specificity of gene delivery to the target cells
- Induction of a strong immune response

DISADVANTAGES:
- Integration into the host genome
- The presence of a pre-existing response to a vector
(less effectiveness of the vaccine)

https://www.niaid.nih.gov/topics/Flu/Research/vaccineResearch/Pages/Technologies.aspx
mRNA vaccines
Ura et al., 2014, Vaccines 2, 624-641
 Adenoviral Vaccine Vectors:

They are used as a carrier of genes encoding "vaccine" antigens.

Most often, the 1st generation - additional stimulation of the immune system.
Vector-based vaccines induce mainly cell-mediated (T-lymphocyte) immune
response
The problem: people with previous contact with the virus have acquired
immunity to vector proteins - this may reduce the effectiveness of vaccination

Clinical trials: HIV, HCV, Ebola, malaria, SARS-CoV-2

Mechanism of action of adenoviral vectors

T cell

adenoviral
vector

nucleus

muscle cell
T cell CD8+

T cell CD4+ B cell

antibodies

Chmielewska A., Trzonkowski P., Bieńkowska-Szewczyk K., Mrukowicz J. 2020, Medycyna Praktyczna 4/2020
 Vaccine against COVID-19 Janssen Pharmaceutica
- Vector vaccine containing the full S (spike) coding sequence of the SARS-CoV-2 virus
- The vector used is a recombinant human adenovirus type 26 that has been modified to
prevent its replication.
- The vector with the S protein sequence was obtained in genetically modified human
embryonic PER.C6 cells (from the retinal tissue of the human embryo).
- The vector provides delivery of the selected coding sequence to the host cells, where it
is transiently expressed and viral protein S is synthesized.
- The S protein stimulates both neutralizing and functional antibodies
against S, as well as other cellular immune responses
directed against the S antigen
Vector vaccine Sputnik V
• It is based on two human modified adenoviruses:
Ad5 and Ad26
• Not approved for use in Europe
Alternative to human adenovirus = chimpanzee adenovirus

• A large number of viral strains

• Possibility of culturing in cell lines approved by the FDA
• No neutralization by human antibodies
• Potential similar to human adenoviruses

Oxford-AstraZeneca's COVID-19 vaccine is based on chimpanzee adenovirus

 Vaccine against COVID-19 Astra Zeneca

• Based on a recombinant modified

chimpanzee adenoviral vector (ChAdOx1)
• It contains the complete S protein coding
sequence
• The produced protein is in a trimer form
• A vector with protein S coding sequence is
obtained in genetically modified human
embryonic kidney cells (HEK-293)
• It does not need to be stored in the freezer.
 Virus-like particles – VLPs ?
• VLPs are non-infectious particles containing all or some outer virion proteins.
• structures of recombinant proteins morphologically very similar to natural virions
having antigenic properties similar to those of native virus particles.
• Safe - no viral genetic material included
• VLPs induce a strong immune response, both cellular and humoral
• VLPs arise spontaneously during viral infection or can be produced using many
expression systems, including baculovirus, plant and mammalian expression systems
• Over 110 different VLPs have been constructed for viruses from 35 different families
• They are made of one or more structural proteins Virus VLPs

FDA approved – hepatitis B virus, human papilloma virus

VLPs
Induce strong immune response Do not replicate

Non-infectious
Structurally similar to a
viral particle, but without a
genome = safe
Stable
Immunogenic features of a VLP presenting foreign antigens
 Baculovirus Expression Vector System
Bac-to-Bac
¢ high level of foreign gene
expression in comparison to
other eukaryotic systems
¢ post and co-translational
modifications similar to
mammalian expression systems
¢ a possibility of expression more
than one foreign gene
¢ baculovirus can accomodate
about 20kb of foreign DNA
¢ addition of signal sequence can
lead to export of a gene product
out of the infected cell
 Novavax Protein S-based vaccine against COVID-19 produced using
baculovirus

New York Times, 2020

Human papilloma virus HPV:

• Family: Papillomaviridae
• 100 types of this virus, some can cause benign changes in the form of
warts on the skin, and some malignant tumors such as cervical cancer
and penile cancer.
• HPV is the most common sexually transmitted disease
• 470,000 cases of cervical cancer diagnosed each year in the world
• 5,000 deaths each year in the US
 Anti-HPV vaccines:

• CERVARIX (GlaxoSmithKline) – bivalent vaccine, provides protection

against HPV serotypes 16 and 18, is based on VLPs obtained in insect
cells made of the L1 envelope protein
• GARDASIL (Merck) – quadrivalent vaccine, provides protection against
serotypes 6, 11, 16 and 18, based on VLPs obtained in yeast cells
• Effective when administered prior to first HPV infection
N-glycosylation process
Differences of N-glycosylation process in insect and
mammalian cells:

Mammalian cells Insect cells

Mammalian Expression Vector System
Nonconventional expression system- Leishmania tarentolae

• Protozoan parasite of gecko

• It is not pathogenic to mammalians and fully
approved for use in biosafety level 1 (S1)
laboratories
• Cheap and easy chandling system
• High expression-success rates with yields of up
to 500 mg per litre of culture
• minimal nutrition requirement
• Full range of Post-Translational Modifications

Post-translational modyfications

jennabioscience.com mammalian- proteolytic

phosphorylation processing oligomerisation
type
acetylation
N-glycosylation
Advantages of LEXSY:
With LEXSY – in six weeks from gene to protein:
Facts about new antiviral vaccines:

• Under the new policy of the European Union, only vaccines that allow for
differentiating infected from vaccinated animals will be allowed to enter the
market (DIVA) (this does not apply to humans)
• Only recombinant vaccines fulfill this principle
• Vaccines based on VLPs are among the most effective recombinant vaccines for
humans.
What is the antigen in DIVA vaccines?
• They consist of two components: a vaccine and a diagnostic test
• Vaccine - a viral antigen that stimulates the immune response
• A diagnostic test is based on a different antigen of the same virus that
is not a component of the vaccine.
• If the antigen used in the test is present in the animal, it indicates an
infection.
Vaccine bioprocessing overview
Discovery
Antigen design
Process Development
Antigen production in lab scale
Production proces
Manufacturing
Assay development development
Adjuvant GMP
Scaling up to pilot scale
Administration method Same immunological
GLP/GMP
properties
Immunology in animal model
Recombinant vaccine antigen production process

Antigen Upstream Downstream Formulation Special Process

(pilot scale and
manufacturing)

Subunit Fermentation Remove cell debris, Addtion of Lyophilization (if

(E.coli, yeast) protein adjuvants, needed)
VLPs/NPs Virus culture (insect Remove nucleic stabilizers
cells) acid
Mammalian cell Concentration of
culture the purified
antygen
Antiviral therapeutics
Antiviral drugs - compounds with the ability to prevent the spread
of viral infection

Antivirals can be divided into:

- virus-targeting antivirals - VTAs
- host-targeting antivirals – HTAs

Antiviral drugs can affect the following viral processes:

- adsorption of viruses to the host cells by binding to a specific receptor;
- penetration - entry of the virus into the host cell;
- release of genetic material;
- production of early proteins needed for viral genome replication
- genome replication - different mechanisms depending on the type of virus
- production of late proteins - based on newly created genomes
- assembly of virions - formation of nucleocapsids
- release of viruses from the host cell
Lou i wsp., 2014, Trends in Pharmacological Sciences, 35: 86-102
"Current Issues in Molecular Virology - Viral Genetics and
Biotechnological Applications”, DOI: 10.5772/56866
Drug life cycle – from idea to the
market:
• 12–15 years – time from identification of a new molecule to drug
registration
• the research and development process for one drug requires 700,874
hours of work (according to data from Roche)
• requires 6587 experiments
• requires the employment of 423 researchers
• the drug must undergo comprehensive in vitro laboratory tests (basic
research), toxicological tests on experimental animals (pre-clinical
studies) and three phases of clinical trials with humans
• 1 in 250 substances successfully pass all stages of research
 Pre-clinical in vitro studies:
• last from 5 to 8 years
• CC50 (cytotoxic concentration 50%) – compound concentration that
reduced cell viability by 50%

• IC50 (inhibitory concentration 50%) - compound concentration

required to inhibit the virus replication by 50%.

• Therapeutic index - S.I. (CC50/IC50)

 Pre-clinical in vivo studies:
• Research on experimental animals
• Determination of toxicity (acute and chronic)
• Determination of the method of absorption, metabolism and secretion of
the compound in the body
• Determination of side effects (teratogenesis, oncogenesis, mutagenesis)
Registration procedure for medicinal products:
Registration procedure for medicinal products:
• The registration process takes 90 - 210 days
• Office for Registration of Medicinal Products, Medical Devices and
Biocidal Products in Poland
• Registration process based on: national, centralized, mutual
recognition or decentralized procedure
Genetic changes in the genome of viruses:
• natural sequence differences of organisms of one species
• mutations are the source of genetic variation (point mutations, deletions,
insertions), phenomenon of recombination and reassortment
• genetic changes can cause the emergence of new sequence variants

emergence of more virulent viral strains (adaptation to new hosts,

emergence of diseases with previously unknown clinical course)
emergence of drug-resistant strains

no diagnostic methods or preventive programs

Viruses characterized by high variability:
- influenza virus (Orthomyxoviridae)
- hepatitis C virus (HCV, Flaviviridae),
- human immunodeficiency virus (HIV, Retroviridae),
- Dengue virus
- West Nile virus
- Zika virus
- SARS, MERS, SARS-CoV-2
- noroviruses (Caliciviridae).
Genetic variability especially in RNA viruses
RNA polymerase - no 3’ → 5 exonuclease activity
(10-2 - 10-6 mutations/nucleotide = 100 errors/104 nucleotides)

SUBSTITUTIONS - changing one base pair to another:

80% transitions (change of purine to another purine or pyrimidine to
another pyrimidine, e.g. G to A or C to T)
20% transversions (change of purine base to pyrimidine base or vice
versa) ==== change of the encoded amino acid, introduction of a stop
codon.
human immunodeficiency
virus
human immunodeficiency virus
= enveloped virus from lentiviruses, Family Retroviridae
= attacks CD4 helper T cells of the immune system
=there are about 40 million virus carriers in the world
=genome - two identical copies of single-stranded RNA –
high genetic variability
=no vaccine available
=no effective method of eliminating the virus from the body available
=some antiviral therapies available
(able to reduce the viral genom to undetectable levels)
Anti-retrovirus drugs:
• Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs),
• non-nucleoside reverse transcriptase inhibitors (NNRTIs),
• protease inhibitors (PIs),
• entry inhibitors,
• integrase inhibitors.
Nucleoside/nucleotide reverse transcriptase inhibitors
(NRTIs):
• inhibit reverse transcription – the conversion of RNA to DNA - NRTIs are chain terminators
• Examples of NRTIs include: zidovudine, abacavir, lamivudine, emtricitabine, and of NtRTIs –
tenofovir and adefovir

Non-nucleoside reverse-transcriptase inhibitors (NNRTIs):

• binding to an allosteric site of the reverse transcriptase
• Examples: nevirapine, efavirenz, etravirine and rilpivirine.

Integrase inhibitors:
• inhibit the viral enzyme integrase, which is responsible for integration of viral DNA into the DNA of
the infected cel
• Examples: raltegravir, elvitegravir, dolutegravir, bictegravir, and cabotegravir.
Protease inhibitors (PI):
• block the viral protease enzyme necessary to produce mature
virions upon budding from the host membranę
• prevent the cleavage of gag and gag/pol precursor proteins
• Examples: lopinavir, indinavir, nelfinavir, amprenavir, ritonavir,
darunavir and atazanavir.

Entry inhibitors (or fusion inhibitors):

• interfere with binding, fusion and entry of HIV-1 to the host cell by
blocking one of several targets.
• Examples: maraviroc and enfuvirtide
Highly Active Antiretroviral Therapy
• The use of multiple drugs that act on different viral targets is known
as highly active antiretroviral therapy (HAART).
• HAART decreases the patient's total burden of HIV, maintains function of
the immune system, and prevents opportunistic infections that often lead
to death
• HAART prevents the transmission of HIVbetween serodiscordant same sex and
opposite sex partners so long as the HIV-positive partner maintains an
undetectable viral load
• Two drugs from NRTI or one from NNRTI and PI
• Expensive therapy, accompanied by various side effects, mainly: vomiting,
diarrhea, anemia, hepatitis, etc.
• It causes the HAND syndrome - a spectrum of neurocognitive
disorders - HIV-associated neurocognitive disorders (HAND)
Hepatitis C virus
 Hepatitis C virus

• Family: Flaviviridae
• Genus: Hepacivirus
• small (55–65 nm in size), enveloped, positive-
sense single-stranded RNA virus.
• 7 genotypes
• the hepatitis C virus is the cause of hepatitis C and
some cancers such as liver cancer (hepatocellular
carcinoma) and lymphomas in humans.
• 180 million people infected worldwide
• every year 350,000 people die due to the diseases
related to HCV infection
• No vaccine available
Two-drug treatment:
• pegylated interferon (PEG-IFN) in combination with ribavirin (RBV)
• duration: genotype 2 and 3 - 24 weeks, genotype 1 and 4 - 48 weeks
• Ribavirin:
- synthetic nucleoside (guanosine) analog
- stimulates a cellular immune response
- inhibits HCV replication by inhibiting the cellular enzyme inosine monophosphate
dehydrogenase (IMPDH) leading to a reduction in the pool of guanosine
triphosphate - the main substrate for the RNA synthesis
- modifies the activity of RNA-dependent polymerase
- improves interferon activity by stimulating ISGs genes (interferon stimulated
genes)
- induces mutagenesis within the genetic material of the virus
- The therapy is ineffective, expensive, causing many side effects
Hepatitis C is treated using:
- direct-acting antiviral (DAA)
- host-targeting agents

Au i wsp., 2014, Clin Pharmacol Ther 2014; 95:78

 Serine protease inhibitors NS3/4A:
• The NS3/4A serine protease catalyzes the hydrolysis of the HCV
polyprotein, cleaves the connections between NS3/NS4A, NS4A/NS4B,
NS4B/NS5A and NS5A/NS5B proteins
• First generation drugs – boceprevir and telaprevir
• Used only in combination therapy with IFN and RBV
• Approved for use in patients infected with HCV genotype 1
• They improve the effectiveness of two-drug treatment, shorten the
duration of therapy, but cause additional side effects
• They induce the formation of drug-resistant strains
Other serine protease inhibitors :
• Simeprevir - a second generation inhibitor, used to treat patients
infected with genotype 1, used orally only once a day, approved for
therapy in combination with PEG - IFN + RBV.
• Grazoprevir – approved for use in combination therapy with elbasvir,
an inhibitor of the NS5A protein that is part of the replication
complex
NS5B polymerase inhibitors:
• Two groups of compounds: nucleoside/nucleotide analogs and non-
nucleoside polymerase inhibitors
• Nucleotide/nucleoside analogs mimic the substrates for the
polymerase, and when incorporated, replication is terminated.
Polymerase active site - highly conserved = activity against different
genotypes.
• Non-nucloside polymerase inhibitors bind to one of the four allosteric
sites of NS5B, which impairs the enzyme activity, active mainly against
HCV genotype 1, induce drug-resistant variants
• Used in three-drug systems with PEG-IFN+RBV, as well as in multi-
component therapy excluding PEG-IFN.
Nucleotide/nucleoside analogs:
Sofosbuvir - a prodrug, converted into an intermediate form of the
drug only in vivo, in the liver turns into a pharmacologically active form
It is incorporated by the polymerase into the newly formed genetic
material, causing chain termination.
Approved for the treatment of HCV genotypes 1-6.
First approved two-drug based therapy - sofosbuvir + RBV without
interferon - the first oral anti-HCV therapy.
Advantages: High efficiency, low toxicity of therapy, high threshold of
drug resistance
Disadvantages: high cost (12-week cycle = 100 000 euro)
NS5A inhibitors:
• NS5A protein – the role in the replication and assembly of progeny virions,
although the exact molecular mechanism of NS5A is unknown =====
the mechanism of action of NS5A inhibitors is also unknown.
• The NS5A protein inhibitors: ledipasvir, ombitasvir, elbasvir and
daclatasvir.
• They show strong antiviral activity, active against various genotypes, but
induce the formation of drug-resistant variants
• Daclatasvir - used orally mainly in combination therapy with sofosbovir or
other DAAs.
• Ledipasvir – used in combination with sofosbuvir.
anti-HCV available therapies:
• Harvoni (sofosbuvir (NS5B) + ledipasvir (NS5A))
• Epclusa (sofosbuvir (NS5B) + velpatasvir (NS5A)),
• Viekira Pak (ombitasvir (NS5A) + paritaprevir (NS3/4A) + ritonavir
(CYP3A4) + dasabuvir (NS5B)),
• Mavyret (glecaprevir (NS3/4A) + pibrentasvir (NS5A))
• Zepatier (elbasvir (NS5A) + grazoprevir (NS3/4A))

https://www.hepatitisc.uw.edu/page/treatment/drugs