Actuality-2017e

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Milling around the frozen doorstep

while opening the icy sack

with 12 months' puzzles inside,

it is January blowing passing year away

to show in Spring and Summer,

paving the way for Autumn and Winter;

2017, what is brought by?

¤¤¤ MEMO January - December 2017 ¤¤¤ Zika virus (continuation from 2016) ¤¤¤ Nobel Prize 2017 ¤¤¤

In Focus: Zika virus - 2017

According to WHO Situation Report 10-March-2017 (→ compared to WHO Situation Report 13-October-2016):

altogether 84 countries/territories have announced vector-mediated Zika transmission.

→ In October-2016 the number was 73 countries/territories.

64 countries reported established population of vector mosquito, with no infections documented.

13 countries recognised human-to-human transmission of Zika infection.

→ In October-2016 the number was 12 countries/territories.

31 countries/territories reported Zika infection related microcephaly and pathological disorders of the central nervous system.

→ In October-2016 the number was 22 countries/territories.

23 countries/territories indicated enhanced frequency of Guillain-Barré syndrome (GBS) and/or Zika infection confirmed by laboratory tests in GBS patients.

Zika virus specificities known so far (for history see → 2016/Actuality) :

[Smartt Ch.T.et al. (+11)(2017): Evidence of Zika Virus RNA Fragments in Aedes albopictus (Diptera: Culicidae) Field-Collected Eggs From Camaçari, Bahia, Brazil J Med Entomol tjx058. doi: 10.1093/jme/tjx058]

a/ horizontal (sexual transmission ✔ via blood transfusion ✔ via other body fluids?)

b/ vertical (from infected mother to fetus)

[Basu R., Tumban E. (2016): Zika Virus on a Spreading Spree: what we now know that was unknown in the 1950’s Virol J. 13: 165. PMCID: PMC5053350]

Besides RT-qPCR, a new trend in detecting virus infection is the on site Point-of-Care (POC) detection giving results within an hour, most preferably in saliva samples, with no need for PCR platforms and performed in portable and disposable microfluidic cassettes (reverse transcription + izothermal nucleic acid amplification + colorimetric detection > sensitivity = 5 PFU). Further option in on site POC detection is the use of smartphone application for wireless actuation, for monitoring nucleic acid amplification, for analyses of results.

[Eboigbodin K. et al. (2016): Rapid molecular diagnostic test for Zika virus with low demands on sample preparation and instrumentation Diagnostic Microbiology and Infectious Disease 86: 369–371.]
[Song J. et al. (2016): Instrument-Free Point-of-Care Molecular Detection of Zika Virus Anal Chem. 88: 7289–7294.]
[Priye A. et al. (2017): A smartphone-based diagnostic platform for rapid detection of Zika, chikungunya, and dengue viruses Scientific Reports 7, Article number: 44778 doi:10.1038/srep44778]

Difference between the virus lines regarding kinetics of replication was confirmed by dendritic cells in vitro infected with Zika lines (dendritic cells of innate immune system = professional cells throughout the body dedicated to antigen recognition and presentation, activating among others virus eliminating CD8+ T cells) . As of the observations, Zika-Africa concluded into more serious infection due to its higher replication rate, ultimately leading to induced cell death. It seems that evolutionary advantage is provided to Zika-Asia by its lower replication kinetics with no induced cell death in the infected cells.
Upon in vitro virus infection only slight dendritic cell activation resulted; the inhibition of Type I Interferon (IFN1) translation helped virus replication evade dendritic cell surveillance. Still, signalling pathways of receptor family RLR (RIG-1-like receptors) are left stimulatable bearing potential for further antiviral actions.
[Bowen J.R., Quicke K.M., Maddur M.S., O’Neal J.T., McDonald C.E., Fedorova N.B., Puri V., Shabman R.S., Pulendran B., Suthar M.S. (2017): Zika Virus Antagonizes Type I Interferon Responses during Infection of Human Dendritic Cells PLOS Pathogens https://doi.org/10.1371/journal.ppat.1006164]
[Yueh-Ming Loo and Michael Gale, Jr. (2011): Immune signaling by RIG-I-like receptors Immunity 34: 680–692.]

[Bayless N.L. et al. (2016): Zika Virus Infection Induces Cranial Neural Crest Cells to Produce Cytokines at Levels Detrimental for Neurogenesis Cell Host & Microbe 20: 423-428.]

[Li H. et al. (2016): Zika Virus Infects Neural Progenitors in the Adult Mouse Brain and Alters Proliferation Cell Stem Cell 19: 593–598.]

[Nowakowski T.J. et al. (2016): Expression Analysis Highlights AXL as a Candidate Zika Virus Entry Receptor in Neural Stem Cells Cell Stem Cell 18: 591-596.]
[Chavali P.L. et al. (+15) (2017): Neurodevelopmental protein Musashi-1 interacts with the Zika genome and promotes viral replication Science 357: 83-88.]
[Ling Yuan et al (+22) (2017): A single mutation in the prM protein of Zika virus contributes to fetal microcephaly Science DOI: 10.1126/science.aam7120]

A further step in experimental approaches is the establishment of a murine model for detailed analysis of neurocognitive deficiencies caused by members of the Flavivirus genus. The first series of observations were collected by infection with flavivirus West Nile Virus (WNV).
→ In mice with acute infection and also in mice after recovery, presynaptic loss i.e. degradation of presynapses with no harm to the axon could be detected. In the hippocampus area of the brain in WNV-infected mice, presynaptic degradation was mediated by the presence of complement cascade initiator C1qa + complement C3 + microglia cells with cell surface C3aR receptors recognizing complement C3 degraded products + microglial phagocytosis machinery (cellular compartments, enzymes, coding genes).
An important message from the observation above: loss of presynapses could also be observed in hippocampus of infected animal lacking mature B lymphocytes. This finding highlights complement functions separate from mature B cell effector mechanisms, in flavivirus infected organism.

[Vasek M.J. et al. (+21)(2016): A complement–microglial axis drives synapse loss during virus-induced memory impairment Nature 534: 538-543. LETTER doi:10.1038/nature18283]

Questions raised

1. Main and auxiliary reasons of Zika vírus

spread,
biogeographical variations,
infection with increased severity of syndromes along with in later epidemics,

from the biological, physical, meteorological, geological, social point of view? Some points have already been discussed (for details → 2016/Actuality).

In broader sense: which of biological, epidemiological, socioeconomical considerations (population size, variations in geno- and phenotypes, population age pyramid, norms and motivations in society, traditions ...) and which of mathematical models (statistical, machine-learning, compartmental-SIR, -SIS, -MSIR, -SEIR, MSEIR, -SEIS, -MSEIS ...) provide help sufficient for forecasting and managing the cyclic emergence-retreat-reappearance of epidemics associated with transmission of infective agents within and inter populations, like in case of Zika virus?

[Neil M. Ferguson et al (+7) (2016): Countering the Zika epidemic in Latin America Science 353: 353-354.]

[Siettos C.I., Russo L. (2013): Mathematical modeling of infectious disease dynamics Virulence 4: 295–306.]
[Kuniyoshi M.L.G., dos Santos F.L.P. (2017): Mathematical modelling of vector-borne diseases and insecticide resistance evolution J Venom Anim Toxins Incl Trop Dis. 23: 34. PMCID: PMC5501426]
Bonyah E.,Khan M.A.,Okosun K.O.,Islam S. (2017): A theoretical model for Zika virus transmission PLoS One. 12(10): e0185540. PMCID: PMC5627930

2. Observations on other members of the Flavivirus genus (Dengue virus/DENV, West Nile virus/WNV, yellow fever virus/YFV, Japanese encephalitis virus/JEV, tick-borne encephalitis virus/TBE) if adaptable in Zika virus research?

3. Host immune history and status, preceding Flavivirus infection (DENV, WNV...) with poor or non-neutralizing antibodies left behind, their aggravating impact on the outcome of the antigenically close Zika infection: chances for ADE (Antibody-Dependent Enhancement) and for OAS (original Antigenic sin)?

Related Experimental Approach

Anti-DENV human monoclonal antibody + Zika virus administered to in vitro myeloid U937 cell line ⇒ determination of virus titer in culture supernatant, FACS analysis of infected culture cells ⇒ in result, observation of Zika-ADE in vitro.

[Dejnirattisai W. et al.(2016): Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus Nat. Immunol. 17: 1102-1108.]

Note

anti-DENV antibodies provoking Zika-ADE are poor neutralizing antibodies.
the timing of Zika infection following DENV infection is crucial since protection against or enhancement of disease by crossreacting antibodies can change with time.

Related Experimental Approach
Anti-DENV human plasma OR anti-WNV human plasma OR purified plasma IgG administered to in vitro myeloid K562 cell line then infected with Zika virus ⇒ in result, observation of Zika-ADE in vitro.

AND

Anti-DENV human plasma OR anti-WNV human plasma OR purified plasma IgG injected into mice then infected with Zika virus ⇒ in result, observation of Zika-ADE in vivo.

[Bardina S.V. et al.(2017): Enhancement of Zika virus pathogenesis by preexisting antiflavivirus immunity Science 356: 175-180.]

Note

ADE was observed at low concentrations of immune plasma and low titers of antibodies. ADE was accompanied by fever (≥ 38 ^oC) and intense viremia in the testes and the spinal cord.
Compared to anti-DENV antibodies, anti-WNV antibodies were less potent in Zika disease enhancement.

Preceding flavivirus (DENV/WNV/...) history followed by Zika infection: chance for OAS?

[Mee Sook Park et al.(2016): Original Antigenic Sin Response to RNA Viruses and Antiviral Immunity Immune Netw. 16: 261-270.]

The immunbiological basis of the phenomenon is the rapid multiplication of viruses and the accompanying diverse antigenicity.
Interpretation of the phenomenon: ability of the first antigen variant to determine - imprint - immune response.

Originally, the phenomenon - immunological imprinting - was observed in influenza virus infection, when the first encounter of the host with a specific virus strain harboring characteristic antigen(s) - variant Ag1 - determined the production of antibodies specific to variant Ag1 even when reinfection happened with the same strain though harboring different antigen(s) - variant Ag2 -.

Preceding non-Flavivirus infection, does it have an impact on the outcome of Zika infection?

4. Development of Zika-selective serodiagnostics OR sero-genodiagnostics?

Detection of Zika infection

- diagnostic windows according to present day knowledge -

Serum sample: few days before and after onset of symptoms.
Whole blood sample: viremia within 2 months following onset of symptoms (left for confirmation).
IgM antibody titer: from 4-7 days to appr. 12 weeks after onset of symptoms.
Neutralizing antibodies: persistence even for years.
Urine sample: higher than in serum virus titer, 7-14 (or more?) days after onset of symptoms.
Saliva sample: higher than in serum virus titer, diagnostic window comparable to that of urine EXCEPT inconsistency of saliva samples.

Marie Louise Landry and Kirsten St. George (2017): Laboratory Diagnosis of Zika Virus Infection Archives of Pathology & Laboratory Medicine 141: 60-67.

5. Preventing epidemics AND support to embryogenesis by new concepts in vaccine design?

[Vidya Mangala Prasad, Andrew S Miller, Thomas Klose, Devika Sirohi, Geeta Buda, Wen Jiang, Richard J Kuhn & Michael G Rossmann (2017): Structure of the immature Zika virus at 9 Å resolution Nature Structural & Molecular Biology doi:10.1038/nsmb.3352 Published online 09 January 2017 ]

The cryo-electronmicroscopic studies have disclosed distinctive features between mature and immature Zika virions hence, the virus map obtained by the observations has later led to diverse solutions in vaccine development.

1. Features of the mature Zika virion

Expression of virus surface 'E' (envelope) protein dimers; among them 'M' (membrane) proteins have hidden conformation ('E' and 'M' are transmembrane proteins).
'E' protein/glycoprotein domains and functions (DI-DII-DIII):

* adhesion of virus to target cells, docking on cell surface receptors (DIII);
* dimerization (DII);
* virus surface+target cell membrane fusion (DII distal hydrophobic, conserved loop);
* DII domain regions flexibly held-together;
* DI domain interposed between DII domain regions.

'E' glycoprotein: main target of neutralizing antibodies.

2. Features of the immature Zika virion

In its intracellular phase of lifecycle, Zika virus particles having assembled in the endoplasmic reticulum of the infected cell move towards the trans-Golgi network (TGN) where the low pH and the proteolytic enzymes provide conditions for further steps in virus maturation (> cleavage of immature virions' polyproteins by furin type enzyme proprotein convertase/serine protease > separation of structural from nonstructural proteins, a process similar to that of Dengue vírus, of Ebola virus, of HIV, of Influenza vírus, under maturation).

Virus surface trimers composed of 'E' (envelope) glycoprotein and 'prM' (precursor membrane) protein.
'E' glycoprotein domain DII: distal hydrophobic conserved loop is hidden (inactive) by 'pr' domain of 'prM' protein (> 'prM' acting here as chaperon to assist 'E' structural formation).
Flavivirus under proteolytic maturation: following cleavage of soluble 'pr' domains from 'prM' proteins, mature virions leave the cell.

[Stadler, K., Allison, S.L., Schalich, J. & Heinz, F.X. (1997): Proteolytic activation of tick-borne encephalitis virus by furin. J. Virol. 71: 8475–8481.]
[Yu, I.-M. et al (2008):. Structure of the immature dengue virus at low pH primes proteolytic maturation. Science 319: 1834–1837.]

Cleavage of 'pr' domain from 'prM' protein can be performed completely, partially, or in fact, can be missing ⇒ viruses released from the infected cell are heterogeneous populations of mature, partially mature and immature virions.

[Poonsook Keelapang et al.(2004): Alterations of pr-M Cleavage and Virus Export in pr-M Junction Chimeric Dengue Viruses J Virol. 78: 2367–2381.]

[Theodore C. Pierson and Michael S. Diamond (2012): Degrees of maturity: The complex structure and biology of flaviviruses Curr Opin Virol. 2: 168–175.]

Virus surface trimers kept together by stable conformational interactions ⇒ virus surface trimer to dimer rearrangement is slow and cumbersome along with maturation.

Unique to Zika virus: capsid protein like densities found on the inner surface of virus membrane > a feature missing in mature virions. Manifestation of genome protection whilst virus maturation?

In effect, features detailed above provide basis for the emergence of heterogeneous virus populations rich in immature forms released from the infected cells, a process ending up in:
⇒ heterogeneity of virus coat antigens ⇒ conformational hindrance of, or, completely missing mature virus surface consensus antigens ⇒ partial cleavage of 'prM' protein giving access to immature surface epitopes ⇒ high amount of neutralizing antibodies produced against more diverse epitopes ⇒ besides traditional solutions in vaccination (e.g. immunization with inactivated virus) new concepts are put into practice.

One of the new concepts is shown in the elucidative studies of Pardi et al. (2017):
[Pardi N. et al. (+36)(2017): Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination Nature 543: 248–251.]

The presence of modified nucleoside m1Ψ in mRNA give rise to enhancement in translation, decrease in mRNA immunogenicity, and increase in mRNA stability.

[Andriesa O. et al.(2015): N1-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice

Journal of Controlled Release 217: 337–344.]

Some points in the technique:

*formulation of vaccine candidate mRNA in lipid nanoparticles.

*'prM'-'E' coexpressing mRNA > translation >>> synthesis of subviral -immature- particles.

*Immunization: 1x intradermal injection

Preclinical observations with vaccine candidate mRNA - experiments in Rodents

In C57BL/6 mice (receiving 1x30 µg in intradermal injection) the level of serum IgG specific for 'E' protein reached its maximum 8 weeks after immunization and was kept by weeks 8-20.

Features of anti-Zika virus neutralizing antibodies (nAB)

A/ The plaque reduction neutralization test₅₀(= PRNT₅₀ nAB assay) showed peak titer by week 16 after immunization, the same was observed until week 20.

B/ Zika virus reporter particle assay (RVP nAB assay) titers were at maximum by week 8 after immunization, and reduced to less than a half by week 20.

C/ Upon infection with heterologous Zika strain (Puerto Rico 2015 strain PRVABC59 / intravenous injection of 200 PFU) on week 2 and 20 after immunization:

* following infection on week 2 after immunization, in 8 of 9 control animals viremia was detected on third day (median 14.000 copies of virus RNA/ml blood).

No viremia was detected in 9 of 9 animals immunized with Zika 'prM-E' mRNA.

* following infection on week 20 after immunization, in 5 of 5 control animals viremia was detected on third day (median 1200 copies of virus RNA/ml blood).

On day 3 and 7 after infection, no viremia was detected in 10 of 10 animals immunized with Zika 'prM-E' mRNA.

Preclinical observations with vaccine candidate mRNA - experiments in Primates

In non-human primate Macaca Mulatta (Rhesus) monkeys (intradermal 1x600µg or 1x200µg or 1x50µg injection) given either dose, the level of serum IgG specific for 'E' protein reached its maximum by week 4 after immunization, and reduced to approximately one-third up to week 12.

Features of anti-Zika virus neutralizing antibodies (nAB)

A/ The focus reduction neutralization test (FRNT) showed stable titers by weeks 2-12 after immunization.

B/ Zika virus reporter particle assay (RVP nAB assay) titers increased by more than one and a half fold from week 2 to week 4 after immunization.

C/ In neutralizing antibody assays, lines Zika-Africa and Zika-Asia represented a single serotype, confirming preceding basic observation referred below.

Basic observation on Single Zika Serotype → Dowd K.A. et al. (+13)(2016): Broadly Neutralizing Activity of Zika Virus-Immune Sera Identifies a Single Viral Serotype Cell Reports 16: 1485–1491.

Expectedly, single Zika serotype makes possible the preventive or therapeutic administration of robust and effective antibody enough for the neutralization of heterologous virus strains. Since the synthesis and reactivity of neutralizing antibody in the experiments did not show the feature of dose-dependence, the application of the lowest dose (here: 1x50µg > cca 0.02 mg/ kg) in non-human primates seemed to be reasonable.

D/ Upon infection with heterologous Zika strain (Puerto Rico 2015 strain PRVABC59 /subcutan injection) on week 5 after immunization:

* in 6 of 6 control monkeys viremia was detected on third day (median 7000 copies virus RNA /ml blood).

* in 4 of 5 monkeys immunized with Zika 'prM-E' mRNA (50µg to 3 animals, 200µg to one animal), viremia was not detectable on days 3, 5, 7 post infection (< 50 copies virus RNA/ml blood). In the animal immunized with the highest dose (600µg), slight viremia was detected on day 3 post infection (100 copies virus RNA/ml blood). The result claims for further experiments with larger number of animals involved.

According to Pardi et al. (2017) studies, single low dose intradermal immunization with Zika 'prM-E' mRNA result in protection of mice and monkeys as well. As of the observations, Zika 'prM-E' mRNA vaccine candidate induces robust neutralizing antibody response.

Other Zika vaccine candidates: Larocca R.A. et al. (2016) and Abbink P. et al. (2016) preclinical studies on plasmid DNA vaccine and on inactivated Zika virus vaccine (for the story see → 2016/Actuality).

Taken together: whether conventional or genetically engineered vaccines of the future, the essential requirements are the same:

Safe vaccine
Long-lasting (lifelong ...) protection
Thrifty large scale manufacturing

In line with preclinical results, with regard to deficiencies of present-day knowledge, Zika vaccine candidates are comparable as follows.

Purified Inactivated Virus Vaccine

chance for infection ???
method adaptable for the expression of any selected protein/antigen (not applicable)
coexpression of multiple selected antigens (not applicable)
thrifty large scale manufacturing ✔
low dose two times administration result in protection ✔
chance for preceding flavivirus history to influence operation of vaccine ✔

plasmid DNA Vaccine Coding for Virus Surface Structural Proteins

chance for integration into the host genome ???
method adaptable for the expression of any selected protein/antigen ✔
coexpression of multiple selected antigens ✔
thrifty large scale manufacturing ???
low dose single/two times administration result in protection ✔
chance for preceding flavivirus history to influence operation of vaccine ???

messenger RNA (mRNA) Vaccine Coding for Virus Surface Structural Proteins

nucleotide sequence not integrating into the host genome ✔
method adaptable for the expression of any selected protein/antigen ✔
coexpression of multiple selected antigens ✔
thrifty large scale manufacturing ???
low dose single administration result in protection ✔
chance for preceding flavivirus history to influence operation of vaccine ???

Beyond B cell mediated immune responses...

What do we know about T cell mediated immune responses in Zika infection? - So far so few.

An analysis on Dengvaxia clinical studies was published in July 2017, authored by S.B. Halstead. (Dengvaxia or CYD-TDV is a recombinant live attenuated four-chimeric/tetravalent vaccine comprising of flavivirus Dengue virus 1-4 structural genes inserted into carrier yellow fever 17D vaccine non-structural genes).
Halstead's thought provoking publication gives emphasis on T cell mediated immunity in the elaboration of immune protection against virus infection. More specifically, it underlines T cell importance in durable protection against heterotypic virus reinfection. Further accent is given to pitfalls in the interpretation of preclinical studies.
As for Halstead's view, at preclinical testing of vaccine constructs in study subject, the presence of vaccine induced neutralizing antibody is not sufficient to meet functional criteria. According to his point, for grading vaccine's antiviral efficacy and durability in study subjects reinfected with virus only some weeks after immunization, the presence of anamnestic neutralizing antibody is not a proof of protection since concentration of antibody reduces with time. Adapting Dengue lessons to those of Zika infection, Halstead stresses CD4+/CD8+ T cell reactions activated by viral non-structural proteins as attributes to durable and effective immune protection (lysis of infected cells, synthesis of cytokine cocktail in support of durable and effective B cell responses induced by live attenuated vaccine ...).
[Scott B Halstead (2017): Achieving safe, effective, and durable Zika virus vaccines: lessons from dengue http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(17)30362-6/fulltext]

In line with those above, at present, there are two options in protection against Zika virus.

Algorithm-1

Pillar in antiviral protection is the antibody specific for viral structural proteins to avoid virus enter into further organs, tissues.

(e.g. antibodies specific for Hepatitis A, Hepatitis B, Japanese encephalitis virus, Poliovirus -1, - 2, - 3, Tick-borne encephalitis)

Zika example

* In mice immunized with plasmid DNA (coding for virus surface 'prM-Env' proteins), removal of CD4+ and/or CD8+ T lymphocytes did not result significant changes in efficient protection against Zika infection. [Larocca R.A. et al.(+24) (2016): Vaccine protection against Zika virus from Brazil Nature 536: 474-478.]

Algorithm-2

Pillar in antiviral protection is the T cell population activated by viral non-structural proteins for supporting effective and durable B cell/antibody response.

(e.g. vaccines of Smallpox, Yellow fever, Measles, Mumps, Rubella, Varicella)

Zika example

* Considering the tendency in Zika spread (→ establishing populations in DENV and WNV endemic regions) and the serological cross reactions between members of Flaviviridae family it is supposed, that DENV specific, cross reactive memory T cells recognize Zika virus in DENV endemic regions (example of 'heterologous immunity').

Question remains: CD4+ and CD8+ T cell mediated reactions in Zika infected individuals?

Back to Zika virus structural and functional characteristics...

A report providing clues for answering some of the questions formatted in a paragraph above, has been published recently.
In their report, Annamalai A.S. et al. discuss glycosylation of Zika virus surface 'E' protein, more specific, glycosylation of 'E' protein motif VNDT in context with virus polymorphism influencing immunogenicity, target cell/tissue tropism. Supporting evidence is the finding that 'E' protein motif VNDT could be detected lately in virus isolates of big epidemics, but not in some isolates from Africa.
Motif VNDT of virus surface 'E' protein dimers (see above: features of mature virion) is situated close to the fusion loop and to the interface between dimers so as the N-glycosylation of motif VNDT, the extent of it have impacts on spatial conformation and also on functions such as virus replication, immunogenicity, transmission, or virulence. Deletion of motif VNDT or, replacement of aminoacids within the motif, both, had no significant effects on virus replication either in vivo (in experimerntal animals) or in vitro (in cell cultures).
Compared to wild type Zika virus, deletion/replacement virus mutants showed significant decrease in their pathogenicity observed mainly at targeting the central nervous system.
[Annamalai A.S. et al (+11) (2017): Zika Virus Encoding Non-Glycosylated Envelope Protein is Attenuated and Defective in Neuroinvasion J. Virol. 91: e01348-17 doi:10.1128/JVI.01348-17]

Concluding question: glycan mutant Zika virus adhesion to or membrane fusion with brain microvascular endothelial cells further, choroid plexus epithelial cells, are modified, maybe failed?

Zika vaccine constructs in clinical trials

Purified inactivated Zika virus ........ conventional active immunization

1. Clinical Trial NCT02963909 / situation on 22-11-2017: 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
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

Single center, randomized, double blind (study subjects and investigators), placebo controlled three armed Phase 1 clinical trial to assess the results of purified inactivated Zika virus vaccine (ZPIV) administered i.m. in two doses 28 days apart to subjects without flavivirus history (flavivírus naif + ZPIV) and to subjects with flavivirus history (flavivírus JEV + ZPIV / flavivírus YFV + ZPIV). A subgroup selected from the study arms are given a third dose of ZPIV.
Primary goal: safety and efficacy of ZPIV administered in two doses to subjects above. Safety and efficacy of the third ZPIV dose.

2. Clinical Trial NCT03008122 / situation on 22-11-2017: 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
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

Single center, randomized, double blind (study subjects and investigators), placebo controlled, dose de-escalating (two experimental doses) Phase 1 clinical trial to evaluate safety and efficacy of purified inactivated Zika virus vaccine (ZPIV) given i.m. in two administration schedule 28 days apart.

3. Clinical Trial NCT02952833 / situation on 22-11-2017: recruiting

ZIKA Vaccine in Naive Subjects
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

Single center, randomized, double blind (study subjects and investigators), placebo controlled dose reducing (three experimental doses) Phase 1 clinical trial for the assessment of safety and efficacy of purified inactivated Zika virus vaccine (ZPIV) given in two i.m. administration 28 days apart, to individuals with no preceding flavivirus history.

Plasmid DNA vaccine coding for virus surface structural proteins 'prM-E' or 'M-E'

1. Clinical Trial NCT02809443 / situation on 22-11-2017: active

Study of GLS-5700 in Healthy Volunteers
Sponsor + Cooperator: GeneOne Life Science, Inc. + Inovio Pharmaceuticals

Multicenter, Phase 1 clinical trial for the follow-up of safety, tolerability, immunogenicity of GLS-5700 vaccine in healthy individuals.
GLS-5700: plasmid with DNA content coding for Zika virus prM (precursor membrane) and E (envelope) transmembrane proteins.

2. Clinical Trial NCT02887482 / situation on 22-11-2017: active

Study of GLS-5700 in Dengue Virus Seropositive Adults

Sponsor + Cooperator: GeneOne Life Science, Inc. + Inovio Pharmaceuticals

Multicenter, randomized, double blind Phase 1 clinical trial for the follow-up of safety, tolerability, immunogenicity of GLS-5700 vaccine in subjects seropositive for Dengue virus.
GLS-5700: plasmid with DNA content coding for Zika virus prM (precursor membrane) and E (envelope) transmembrane proteins.

3. Clinical Trial NCT02840487 / situation on 22-11-2017: active

Safety and Immunogenicity of a Zika Virus DNA Vaccine, VRC-ZKADNA085-00-VP, in Healthy Adults
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

Multicenter, randomized Phase 1/1b clinical trial for the follow-up of safety, tolerability and immunogenicity of Zika virus DNA vaccine (VRC-ZKADNA085-00-VP) administered in four i.m. dosing designs.
Vaccine: circular DNA plasmid coding for Zika virus strain H/PF/2013 prM (precursor membrane) and E (envelope) transmembrane proteins.

4. Clinical Trial NCT02996461 / situation on 22-11-2017: 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
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

Single center, randomized Phase 1 clinical trial to study safety, tolerability, immunogenicity of Zika virus DNA vaccine (VRC-ZKADNA090-00-VP) administered i.m. in three dosing designs and manners.
Vakcina: circular DNA plasmid coding for Zika virus strain H/PF/2013 prM (precursor membrane) and E (envelope) transmembrane proteins.

messenger RNA (mRNA) vaccine coding for virus surface structural proteins

1. Clinical Trial NCT03014089 / situation on 22-11-2017: recruiting

Safety, Tolerability, and Immunogenicity of mRNA-1325 in Healthy Adult Subjects

Sponsor + Cooperator: Moderna Therapeutics + Biomedical Advanced Research and Development Authority

Multicenter, randomized, placebo controlled, dose finding Phase 1/2 clinical trial for the follow-up of safety, immunogenicity of Zika mRNA 1325 vaccine in healthy individuals of non-endemic Zika free regions.

Source: ClinicalTrials.gov

A critical summary of Zika vaccine development racing with time, is at reach here:

[Chris Morrison(2016): DNA vaccines against Zika virus speed into clinical trials Nature Reviews Drug Discovery 15: 521–522.]

An alternative in managing and preventing Zika infection

Anti-Zika Chemotherapy

[Pascoalino B.S. et al.(2016): Zika antiviral chemotherapy: identification of drugs and promising starting points for drug discovery from an FDA-approved library PMCID: PMC5112578]

The novelty disclosed in this remarkable publication is the robust pharmacological testing of FDA-approved chemicals in Zika virus (KX197192.1/Pernambuco-Brazil/2015) infected in vitro cells (human hepatoma cells=Huh7; Aedes albopictus cells=C636; HB-112 mouse hybridoma cells and the ascites produced) by HCS/High Content Screening, a method comprising pharmacological and imaging technics. An advantage of the method covering 72h incubation of the virus with the cells in vitro, is the combined observation and evaluation of viruses and chemicals in action with conventional morphological parameters included (→ virus cellular localization by indirect immunofluorescence using anti-'E' monoclonal antibodies). For antiviral reference compound, recombinant IFNα2A (interferon) was introduced.
Among the 725 compounds screened, due to criteria of selectivity, low toxicity and maximum antiviral effects, five compounds were found appropriate for further studies. The chemically and pharmacologically distinct five compounds are:
→ Lovastatin (lipid lowering substance)
→ 5-Fluorouracil (antineoplastic substance; irreversible inhibition of thymidylate synthase)
→ 6-Azauridine (antineoplastic and anti-psoriasis substance, broad spectrum antimetabolite, inhibition of DNA virus/RNA virus replication)
→ Palonosetron (5-HT₃ antagonist, antiemetic drug)
→ Kitasamycin (macrolide antibiotic).

Nobel Prize 2017

Physiology and Medicine Discoveries of molecular mechanisms controlling biological clock, circadian rhythm (circadian oscillations).	J.C. Hall, M. Rosbash, M.W. Young	References (more: www.nobelprize.org ; Pubmed) Zehring W.A., Wheeler D.A., Reddy P., Konopka R.J., Kyriacou C.P., Rosbash M., and Hall J.C. (1984): P-element transformation with period locus DNA restores rhythmicity to mutant, arrhythmic Drosophila melanogaster Cell 39: 369-376. Siwicki K.K., Eastman C., Petersen G., Rosbash M., and Hall J.C. (1988): Antibodies to the period gene product of Drosophila reveal diverse tissue distribution and rhythmic changes in the visual system Neuron 1: 141-150. Veleri S., Brandes C., Helfrich-Förster C., Hall J.C., Stanewsky R.A. (2003): A self-sustaining, light-entrainable circadian oscillator in the Drosophila brain. Curr Biol. 13: 1758-1767. Bamne M.N., Ponder C.A., Wood J.A., Mansour H., Frank E., Kupfer D.J., Young M.W., Nimgaonkar V.L.(2013): Application of an ex vivo cellular model of circadian variation for bipolar disorder research: a proof of concept study Bipolar Disord. 15: 694-700. Crane B.R., Young M.W. (2014): Interactive features of proteins composing eukaryotic circadian clocks Annual Rev. Biochem. 83: 191-219. Garaulet D.L., Sun K., Li W., Wen J., Panzarino A.M., O'Neil J.L., Hiesinger P.R., Young M.W., Lai E.C.(2016): miR-124 Regulates Diverse Aspects of Rhythmic Behavior in Drosophila J Neurosci. 36: 3414-3421. Vienne J., Spann R., Guo F., Rosbash M. (2016): Age-Related Reduction of Recovery Sleep and Arousal Threshold in Drosophila Sleep 39: 1613-1624. ........................................................................................................... Scientific Background Discoveries of Molecular Mechanisms Controlling the Circadian Rhythm The Nobel Assembly at Karolinska Institutet
Physics LIGO detector (Laser Interferometer Gravitational-Wave Observatory); direct observation of gravitational waves.	R. Weiss (1/2), B.C. Barish (1/4) K.S. Thorne (1/4)	Reference (more: www.nobelprize.org) Scientific Background on the Nobel Prize in Physics 2017 THE LASER INTERFEROMETER GRAVITATIONAL-WAVE OBSERVATORY AND THE FIRST DIRECT OBSERVATION OF GRAVITATIONAL WAVES The Nobel Committee for Physics
Chemistry Developing cryo-electron microscope: high resolution structure determination of biomolecules in solution.	J. Dubochet J. Frank R. Henderson	Reference (more: www.nobelprize.org) Scientific Background on the Nobel Prize in Chemistry 2017 THE DEVELOPMENT OF CRYO-ELECTRON MICROSCOPY
Literature "... in novels of great emotional force, (he) has uncovered the abyss beneath our illusory sense of connection with the world".	Kazuo Ishiguro	Reference (more: www.nobelprize.org) Biography Film adaptation: The Remains of the Day (1993) 134 min
Peace "... for its work to draw attention to the catastrophic humanitarian consequences of any use of nuclear weapons .... for its ground-breaking efforts to achieve a treaty-based prohibition of such weapons".	-ICAN- International Campaign to Abolish Nuclear Weapons	Reference (more: www.nobelprize.org) Australia - 2017 Global civil campaign -ICAN- against nuclear weapons is founded for calling attention to the catastrophic consequences of their use. The aim of ICAN: treaty-based international ban on use of any nuclear weapons.
Economy "...for his contributions to behavioural economics...".		Reference (more: www.nobelprize.org) Scientific Background on the Sveriges Riksbank Prize in Economic Sciences in Memory of Alfred Nobel 2017 RICHARD H. THALER: INTEGRATING ECONOMICS WITH PSYCHOLOGY The Committee for the Prize in Economic Sciences in Memory of Alfred Nobel

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