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Zika virus    2018-1                                             
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) nave 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: 
  1. immunogenic substance of non-proliferative character,
  2. no chance for interference (ADE) with previous vaccine induced immunity.




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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).  

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Background knowledge

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).

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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.  

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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 g/ml RBV cytotoxicity observed at 100 g/ml concentration proved to be slight*, RBV cytotoxicity observed at 1000 g/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 g/ml  (cytotoxicity*)
[RBV + IFNa]  >  [RBVmono]      if RBV   100 g/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.
CLINICAL  TRIALS
(history:  Actuality 2017/Zika virus/...
... 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 Nave 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. NCT02809443situation 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. NCT03014089situation 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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