BIOTEOMED.ORG
PRECLINICAL
RESEARCH
Albert To et al.
(2018): Recombinant Zika Virus Subunits Are Immunogenic and Efficacious
in Mice January/February
mSphere 3: e00576-17 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760751/
Disclosure of the inventive
platform developed for Flavivirus Dengue
(DENV) and West Nile (WNV),
as adapted for the
preparation of Zika virus (ZIKV) vaccine
candidate; the
preparation of experimental ZIKV vaccine candidate
comprising the
recombinant subunit region of the immunogenic
protein (glycoprotein) expressed on the virus envelope
surface.
For ZIKV, recombinant ectodomain (about
45 kD protein antigen) of virus surface 'E' glycoprotein
was produced in Drosophila melanogaster (fruit fly) stable
transformed S2 (macrophage-like) cell
culture (sterile
filtration of bioreactor S2 medium, purification by affinity
chromatography, SDS-PAGE and Western blot confirmation, sterile
filtration and freezing storage).
The purified
product (Zika virus recombinant subunit antigen)
was bound to microparticle support and formulated with addition of
different adjuvants (2%
Alhydrogel // Imject Alum // CoVaccine HT) or
without adjuvant to
reach final form
of the vaccine candidate. Immunogenicity,
robustness and safety were
investigated
by immunization of experimental mice with distinct
immunological
backgrounds (ZIKV-E recombinant
vaccine candidate injected
in two i.m.
doses into inbred C57BL/6 mice
with Th1-dominant/cellular
immune reactions,
into inbred
BALB/c
mice with Th2-dominant/humoral immune
reactions and, into SW -outbred- mice).
Serological tests were performed two weeks after
vaccination(s).
Results
- The
definite rise in post-immunization IgG antibody titres
including enhanced levels of antigen-binding and
antigen-neutralizing
antibodies both, are explained by adequate steric conformation
of the experimental vaccine (spatial conformation efficiency
in antigen
presentation to immune cells).
- Both
in
Th1-dominant and Th2-dominant inbred mice, the second dose of
vaccine candidate proved uniformly immunogenic and neither of the
various adjuvants added altered the immune response.
- Compared
to Th1-dominant and Th2-dominant inbred mice, outbred wild mice
had lower titers of post-immunization IgG, albeit significant elevation
of it could be observed after
each i.m.
dose of the vaccine candidate. Simultaneous
appearance of antigen
binding and antigen
neutralizing antibodies in the
wild strain too, were detectable upon immunization. In
members of the wild strain, the elevations of IgG titers were
not coherent, it varied with the heterogeneity of the population.
However, for heterogeneous populations
- and human populations
are alike -, dose-response studies are
required for determining the immune dose triggering
optimum seroconversion.
- Protective
and preventive properties of the vaccine candidate were
studied in BALB/c (Th2-dominant)
mice infected with Zika virus (systemic
viraemia)
either after immunization or prior to it. It
was found that pre-immunized animals were fully protected in
post-infection experiments (no anamnestic antibodies were
found!). In
these experiments, the efficiency of the experimental vaccine
increased to
varying degrees according
to the adjuvant
added.
- Passive
Immunization (intravenous
adoptive transfer
of IgG
purified from sera of immunized animals):
anti-viral passive protection of BALB/c
(Th2-dominant) naïve
mice injected with antibodies of high
IgG titer (antigen-binding
AND antigen neutralizing antibodies) proved sufficient in
coping with subsequent Zika infection.
- Benefits
of the recombinant subunit vaccine candidate:
- immunogenic
substance of non-proliferative character,
- no
chance for interference (ADE) with
previous vaccine induced immunity.
▓░▓▒▓░▓▒▓░▓▒▓░▓▒▓░▓░▓▒▓░▓▒▓░▓▒▓░▓▒▓░▓░▓▒▓░▓▒▓░▓▒▓
Pires de Mello C.P. et al.
(2018): Zika
Virus Replication Is Substantially Inhibited by Novel Favipiravir and
Interferon Alpha Combination Regimens
Antimicrob. Agents Chemother. January 2018
vol. 62 no. 1 e01983-17
Authors studied the in
vitro effect of three broad spectrum antiviral
agents,
Favipiravir (FAV), Ribavirin (RBV) and Interferon-alpha
(IFNa) monotherapy and their paired combinations, in Zika
virus (ZIKV) infected Vero cells.
Results were interpreted and evaluated with the help of "mechanism
based mathematical model (MBM)" disclosed in the publication.
Experimental layout
Cercopithecidae/..../Chlorocebus
sp./African green monkey kidney epithelial cells in vitro (Vero
cells in culture).
Infection of
cells with ZIKV.
Treatment:
antiviral agent (mono or combined) in one dose to the cell
culture.
Screening
monotherapy: daily analysis of cell culture supernatants
(through 4
experimental days, virus functional plaque assay, PFU/ml).
Screening
combination therapy: analysis of cell culture supernatants on 3rd day
after treatment (virus
functional plaque assay, PFU/ml).
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
FAV = substituted pyrazine,
pyrimidine analogue prodrug converted into drug in the recipient and
acting as pseudopurine, RNA-dependent RNA polymerase
inhibitor. In replicating RNA
virus genome (Arena-, Ebola, Entero-, non-Zika flavivirus, Hanta-, Influenza, Norovirus) FAV is irregularly incorporated into the new RNA strand in positions
opposite to template C and U (base transitions). The resulting surpass
of replication error threshold leads to lethal mutagenesis, whereby virus population with
incorporated FAV becomes subject to selection.
RBV = guanosine analogue
prodrug with versatile and controversial
mechanism of action. In replicating RNA virus genome RBV is
incorporated into the new RNA strand so that RNA chain
elongation
continues with incorrect base incorporation (base transitions). The result
of transition mutation
is the selection of virions with RBV incorporation (surpassing
replication error threshold) i.e. a decrease in the variability of virus population,
in quasispecies pathogenic fitness will follow. Due to this
latter, virus strategies evading host protective actions turn to be
limited. Further, in RBV
combination therapy, the chance for developing virus
resistance is diminished.
RBV has effects on DNA viruses too, with unexplained mechanism
of action.
IFNa
= interferon alfa, a paracrine/autocrine cytokine produced by
cells in response to virus infection. It exerts its effects by
activating target cell receptors and postreceptor signalling pathways; interferon-stimulated gene expression (ISG)
inhibits virus multiplication by interfering with supporting
nucleic acid and protein synthesis. In virus infection early phase, in the metabolic match
performed between infecting viruses and host innate
immune response elements, virus multiplication is
repressed by IFNa modulating several metabolic pathways (e.g.
cholesterol, polyamine, tryptophan
synthesis).
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
In
patients with chronic infection of hepatitis C, RBV
monotherapy performed weak antiviral effects. Besides, indirect
biochemical effect of RBV is manifested in decreasing serum ALT (alanine transaminase) activity indicating
some histological recovery in the liver.
In RBV+IFNa (pegylated interferon alfa-2a)
combination the interferon-stimulated hepatic gene expression was
potentiated by RBV in patients with chronic infection of hepatitis C .
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
Results
- FAV
monotherapy: time-delayed ZIKV production, followed by dose-dependent
persistent decrease in viral load during the experiment
(4 days).
In
the assay system, FAV-EC50 (50% effective concentration) = 316.6 μM
(49.74 μg/ml). No cytotoxicity was observed in ZIKV-free control
cells at the highest FAV concentrations either.
- IFNa
monotherapy: persistent decrease (through
4 days of the experiments) in ZIKV
production was only achieved at high concentration (10.000
IU/ml). Lower
concentration range (100 IU/ml - 1000 IU/ml)
resulted in transient and unsustainable inhibition of virus
production.
In
the assay system, IFNa-EC50 = 407.8 IU/ml. No
cytotoxicity
was observed in ZIKV-free control cells at the
highest IFNa
concentrations either.
- RBV
monotherapy: the effective
concentration range (100 μg / ml to 1000 μg/ml)
resulted in transient, unsustainable decrease in viral
production. In the assay
system, RBV-EC50
= 121.7 µg/ml.
RBV cytotoxicity observed at 100 µg/ml concentration
proved
to be slight*, RBV cytotoxicity observed at 1000
µg/ml concentration proved to be much stronger*, the latter
partly
explaining RBV antiviral effects.
- Strength of
combitherapy compared to monotherapy for decreasing virus
production/maturation (experimental
day 3 following drug administration):
[FAV
+ IFNa] > [FAVmono]
[FAV +
IFNa] >
[IFNamono]
[FAV +
IFNa] > [RBVmono]
[FAV
+ RBV] ≡
[FAVmono]
[RBV + IFNa] >
[IFNamono]
if RBV ≥
100 µg/ml
(cytotoxicity*)
[RBV
+ IFNa] >
[RBVmono]
if
RBV ≥
100 µg/ml
(cytotoxicity*)
Taken
together: in experimental
anti-Zika therapy
the combination of [FAV + IFNa] proved
to be effective.
Drawbacks:
* FAV-teratogenicity
in experimental animals, FAV-embryotoxicity
*
blood-brain barrier is not passed by IFNa
- Central
idea of the publication is developing a mathematical model for
predicting drugs competent for virus targeting
and optimised
for combination therapy.
The two direct acting antiviral compounds of the experimental
[FAV + RBV] combitherapy act by similar mechanisms. As
for mathematical considerations of the published paper, this
combination
results in significant antagonism in the interpretation of the competitive interactive model. Indeed, in
experiments with [FAV + RBV] combitherapy the resulting inhibition was
similar to that of [FAVmono] treatment.
- The mathematical model introduced is static,
since no consideration is taken on the following:
1.
time-dependent changes in immune reactions accompanying
actions of antiviral drugs,
2.
active substance pharmacokinetics,
3.
intrapopulation variations of the above mentioned 1.-
2.
The
increased
frequency in 2016 of neurotropic
teratogenic effects accompanying ZIKV
infection revealed the
urge for chemotherapeutics adequate for
inhibiting viral
transmission both in horizontal (zoonotic, further,
human-to-human) and in vertical (mother-to-fetus)
ways. So
came Sofosbuvir (SB), an
approved nucleotide analogue antiviral
medication as a choice for anti-Zika experiments. SB
is a pyrimidine nucleotide analogue prodrug, after
metabolism in the recipient it
exerts effects without the need for combination with IFNa (SB
as defective substrate inhibits RNA dependent RNA polymerase in RNA
viruses). In
human therapy SB is
indicated for patients with chronic infection of hepatitis C
virus. According
to experimental results so far, SB is well tolerated
in experimental animals, ZIKV vertical transmission
is
inhibited by
it, hence, the fetus in infected pregnancy can be saved.
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CLINICAL
TRIALS
...
Zika
vaccine constructs ...)
Purified
inactivated Zika virus: conventional active immunization
1. NCT02963909 / situation
on 04-04-2018: Active
A Phase 1, First-in-human,
Double-blinded, Randomized,
Placebo-controlled Trial of a Zika Virus Purified Inactivated Vaccine
(ZPIV) With Alum Adjuvant in Healthy Flavivirus-naive and
Flavivirus-Primed Subjects
Study Start Date: November 1,
2016
Estimated Primary Completion Date: February 1, 2019
2. NCT03008122 / situation
on 04-04-2018: Recruiting
Phase
I, Randomized, Double-blinded, Placebo-Controlled Dose De-escalation
Study to Evaluate Safety and Immunogenicity of Alum Adjuvanted Zika
Virus Purified Inactivated Vaccine (ZPIV) in Adults in a Flavivirus
Endemic Area
Study Start Date: February 24, 2017
Estimated Primary Completion Date: July 18, 2019
Estimated Study Completion Date: January 15, 2020
3. NCT02952833
/ situation
on 04-04-2018: Active
ZIKA Vaccine in Naive Subjects
(Phase 1, Double-blinded,
Placebo-Controlled Study of
the Safety and Immunogenicity of Alum Adjuvanted Zika Virus Purified
Inactivated Vaccine (ZPIV) Administered by the Intramuscular Route in
Flavivirus Naïve Adult Subjects)
Study
Start Date:
October 14, 2016
Estimated Primary Completion Date: December 18, 2018
Estimated Study Completion Date: June 25, 2019 |
Plasmid
DNA vaccine coding for virus surface structural proteins 'prM-E' or
'M-E'
1. NCT02809443 / situation
on 04-04-2018: Active
Study of GLS-5700 in Healthy
Volunteers
Study Start Date: July, 2016
Estimated Primary Completion Date: November, 2017
Estimated Study Completion Date: December, 2017
Preliminary
Report: Tebas P. et al (2017): Safety and
Immunogenicity of an Anti–Zika Virus DNA
Vaccine -Preliminary Report
DOI: 10.1056/NEJMoa1708120
2. NCT02887482 / situation
on 04-04-2018: Active
Study of
GLS-5700 in Dengue Virus Seropositive Adults
Study
Start Date: August, 2016
Estimated Primary Completion Date: October, 2017
Estimated Study Completion Date: June, 2018
3. NCT02840487 / situation
on 04-04-2018: Active
Safety and Immunogenicity of a
Zika Virus DNA Vaccine, VRC-ZKADNA085-00-VP, in Healthy Adults
Study Start Date:
August 2, 2016
Estimated Primary Completion Date:
December 28, 2018
Estimated Study Completion Date:
December 28, 2018
4. NCT02996461 / situation
on 04-04-2018: Active
VRC
320: A Phase I, Randomized Clinical Trial to Evaluate the Safety and
Immunogenicity of a Zika Virus DNA Vaccine, VRC-ZKADNA090-00-VP,
Administered Via Needle and Syringe or Needle-free Injector, PharmaJet,
inHealthy Adults
Study Start Date: December 16, 2016
Estimated Primary Completion Date: December 28, 2018
Estimated Study Completion Date: December 28, 2018
5. NCT03110770 / situation
on 04-04-2018: Recruiting
VRC705: A Zika Virus DNA Vaccine in Healthy Adults and Adolescents (VRC-ZKADNA090-00-VP in Phase 2)
Study Start Date: 29 March, 2017
Estimated Primary Completion Date: January, 2020
Estimated Study Completion Date: January, 2020 |
messenger
RNA (mRNA) Vaccine coding for virus surface structural proteins
1.
NCT03014089 / situation
on March 8, 2018: Active
Safety, Tolerability, and
Immunogenicity of mRNA-1325 in Healthy Adult Subjects
Study Start Date:
December, 2016
Estimated Primary Completion Date:
September, 2018
Estimated Study Completion Date:
September, 2018 |
Source:
ClinicalTrials.gov |
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