MEMO 2016 April-December  ¤¤¤  Nobel Prize 2016  ¤¤¤   Zika Virus 2016  ¤¤¤  HIV/AIDS Virus  2016 


Nobel Prize - 2016  

Details from Alfred Bernhard Nobel's (Stockholm, 21 October 1833 - San Remo, 10 December 1896) Will
(Paris, 27 November 1895)

".... The whole of my remaining realizable estate shall be dealt with in the following way: the capital ..... shall constitute a fund, the interest on which shall be annually distributed in the form of prizes to those who, during the preceding year, shall have conferred the greatest benefit to mankind. The said interest shall be divided into five equal parts, which shall be apportioned as follows: one part to the person ..... within the field of physics; one part to the person ..... (within)  chemical discovery or improvement; one part to the person ..... within the domain of physiology or medicine; one part to the person ..... in the field of literature; and one part to the person ..... for fraternity between nations, for the abolition or reduction of standing armies and for the holding and promotion of peace congresses. The prizes for physics and chemistry shall be awarded by the Swedish Academy of Sciences; that for physiological or medical work by the Caroline Institute in Stockholm; that for literature by the Academy in Stockholm, and that for champions of peace by a committee of five persons to be elected by the Norwegian Storting. It is my express wish that in awarding the prizes no consideration whatever shall be given to the nationality of the candidates, but that the most worthy shall receive the prize, whether he be a Scandinavian or not..."
[source: http://www.nobelprize.org]                                                                                                                           (Emphasis in Italics given by BTM)

It goes to Professor Yoshinori Ohsumi, for his research to reveal the mechanisms and genetic background of autophagy in living cells.
Yoshinori Ohsumi (1945, Fukuoka, Japan) is a cell biologist, received his Ph.D. in 1974 (University of Tokyo). After three years research work in the USA (Rockefeller University/New York)  he returned to his homeland Japan (University of Tokyo), where he established his study group in 1988. Since the year 2009 he is a professor at the 'Tokyo Institute of Technology'.
Basic publications introducing research results:
Takeshige K., Baba M., Tsuboi S., Noda T., Ohsumi, Y. (1992): Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction. Journal of Cell Biology 119: 301-311.
Tsukada M., Ohsumi Y. (1993): Isolation and characterization of autophagy-defective mutants of Saccharomyces cervisiae. FEBS Letters 333: 169-174.
Mizushima N., Noda T., Yoshimori T., Tanaka Y., Ishii T., George M.D., Klionsky D.J., Ohsumi M., Ohsumi, Y. (1998): A protein conjugation system essential for autophagy. Nature 395: 395-398.
Ichimura Y., Kirisako T., Takao T., Satomi Y., Shimonishi Y., Ishihara N., Mizushima N., Tanida I., Kominam, E., Ohsumi M., Noda T. and Ohsumi Y. (2000): A ubiquitin-like system mediates protein lipidation. Nature 408: 488-492.
Source: www.nobelprize.org
It goes to: David Thouless (Prize Share 1/2); F. Duncan M. Haldane (Prize Share 1/4); J. Michael Kosterlitz (Prize Share 1/4), for the "theoretical discoveries of topological phase transitions and topological phases of matter".

David Thouless (1934, Bearsden, Scotland, UK) is a physicist, received his Ph.D. in 1958 (Cornell University). Professional periods: Physicist (Lawrence Berkeley National Laboratory, 1958-1959), Research Fellow (University of Birmingham, 1959-1961), Lecturer (Cambridge University, 1961-1965), Professor of Mathematical Physics (University of Birmingham, 1965-1978), Professor of Applied Sciences (Yale University, 1979-1980), Professor of Physics (University of Washington, 1980-2003), Professor Emeritus (University of Washington, from 2003 on).
F. Duncan M. Haldane (1951, London, UK) is a physicist, at present is a Professor of Physics (Eugene Higgins Professor of Physics, Princeton University, USA). After graduation (St. Paul's School London, Christ's College Cambridge) he turned to the problems in 'condensed matter physics' and contributed to science by his basic discoveries.
J. Michael Kosterlitz (1942, Aberdeen, UK) after graduation (BA - Gonville and Caius College, Cambridge University 1965; MA - ugyanott 1966, PhD. - Oxford University, 1969) until 1981, went on in studies of mathematical physics at several universities (italian, british, american) as research fellow, lecturer, reader. Since 1982 he is Professor of Physics (Brown University).
Basic publications introducing the discoveries:
Kosterlitz J.M., Thouless D.J. (1973): Ordering, metastability and phase transitions in two-dimensional systems. Journal of Physics C: Solid State Physics  6: 1181-1203. (1973)
Thouless D., Kohmoto M., Nightingale M., den Nijs M.(1982):  Quantized Hall Conductance in a Two-Dimensional Periodic Potential. Phys. Rev. Lett. 49: 405-408.
Haldane F.D.M. (1988): Quantum Hall effect without Landau levels: a condensed-matter realization of the parity anomaly. Phys. Rev. Lett. 61: 2015-2018.
Source: www.nobelprize.org; 
It goes to:  Jean-Pierre Sauvage,  Sir J. Fraser Stoddart,  Bernard L. Feringa, "for the design and synthesis of molecular machines".

Jean-Pierre Sauvage (1944, Paris, France) is a chemist working in the field of supramolecular chemistry. He received his Ph.D. in 1971 (University of Strasbourg, France). At present, he is Professor Emeritus professzor (University of Strasbourg) directing a research group (National Center for Scientific Research- CNRS, France).
Sir J. Fraser Stoddart (1942,
Edinburgh, UK)
is a scottish chemist with a working record in supramolecular chemistry. He was given his Ph.D.degree in 1966 (Edinburgh University, UK). Today, he is Professor of Chemistry (Board of Trustees Professor of Chemistry, Northwestern University, Evanston, IL, USA).
Bernard L. Feringa (1951, Barger-Compascuum, the Netherlands) is a synthetic organic chemist. He received his Ph.D. in 1978 (University of Groningen). At present, he is Professor of Organic Chemistry (University of Groningen).

Basic publications introducing the results in the design and synthesis of molecular machines:
Cesario M., Dietrich-Buchecker C.O., Guilhem J., Pascard C., Sauvage J.P. (1985): Molecular structure of catenand and its copper(I) catenate: complete rearrangement of the interlocked macrocyclic ligands by complexation.   
J. Chem. Soc., Chem. Commun. 1985: 244-247.  
Dietrich-Buchecker, Christiane O.,Sauvage J.P. (1989): A Synthetic Molecular Trefoil Knot.  Angewandte Chemie International Edition in English 28(2): 189-192.
Schoevaars A. M., Kruizinga W., Zijlstra R. W. J., Veldman N., Spek A. L., Feringa B.(1997): Toward a switchable molecular rotor. Unexpected dynamic behavior of functionalized overcrowded alkenes.   Journal of Organic Chemistry  62: 4943-4948.
Bravo J.A., Raymo F.M., Stoddart J.F., White A.J.P., Williams D.J.(1998): High Yielding Template-Directed Syntheses of [2]Rotaxanes.  European J. Organic Chemistry  11: 2565-2571.

Source: www.nobelprize.org 

It goes to: Juan Manuel Santos President of Colombia (and the many unknown and known helpers) "for his resolute efforts to bring the country's more than 50-year-long civil war to an end".

Juan Manuel Santos (1951, Bogotá, Colombia) is 32nd President of Colombia since 2010. After studies in economy (University of Kansas) and political sciences (London School of Economics and Political Science) he became deputy director and within two years director of the Colombian 'El Tiempo' journal. He worked as economic advisor (National Federation of Coffee Growers of Colombia), and, as delegate to London (International Coffee Organization). Since 1991 he was Minister of Foreign Trade in Colombia. Later, since 2000 he was Minister of Finance and Public Credit. He was appointed Minister of National Defence in 2006. His many years effort in this latter has led to a peace treaty with guerilla groups fighting in Colombia. However, the recent referendum held over the treaty/the deal, failed. 

Source:  www.nobelprize.org
It goes to: Oliver Hart, Bengt R. Holström, "for their contributions to contract theory".

Oliver Hart (1948, London) completed studies in 'mathematics and science' (1969, BA - King's College, Cambridge) and dropped in economy training (1972, MA - University of Warwick). In the latter he earned his Ph.D. degree (1974, Princeton University). He worked in Cambridge (Churchill College) and London (School of Economics). He has been Professor of Economy since 1993 (Andrew E.Furer Professor of Economics, Harvard  University, Cambridge, MA, USA).
Bengt Holström (1949, Helsinki) finished studies in 'mathematics and science' (BS, University of Helsinki), reached MS level in 1975 (Operations Research, Stanford University), and soon thereafter received his Ph.D. degree (Graduate School of Business, Stanford University). He has been Professor of Economy since 1994 (Paul A. Samuelson Professor of Economics, Massachusetts Institute of Technology, Cambridge, MA, USA).

Basic publications introducing their work:
Hart O., Moore J. (1988): Incomplete Contracts and Renegotiation.   Econometrica 56 (4).
Hart, Oliver, 1995.:  Firms, Contracts, and Financial Structure. New York: Oxford University Press, USA
Fudenberg D., Holmstrom B., Milgrom P. (1990): Short Term Contracts and Long Term Agency Relationships.   Journal of Economic Theory  51: 1-31.

Holmstrom B., Milgrom P.(1991): Multitask Principal-Agent Analyses: Incentive Contracts, Asset Ownership, and Job Design.   Journal of Law, Economics, & Organization  7: 24-52. 


It goes to: Bob Dylan, for "having created new poetic expressions within the great American song tradition".

Bob Dylan (born Robert Allen Zimmerman 1941, Duluth, Minnesota, USA) is a songwriter-and-lyricist. In the 1960s some of his songs became anthems in the Afro-American Civil Rights Movements and also in the protests against America's engagement in the Vietnam war (Blowin' in the Wind, The Times They Are a-Changin). Bob Dylan's more than five decades art work has a blend of style supplied by the american folk, blues, country, gospel, rock and roll music, and the English, Scottish, Irish folk music, intertwined with jazz motifs, too. It is his specialty handling the guitar, the keyboards and the harmonica meanwhile vocal performances. Since 1994 his drawings and paintings have been published in six books.





Part  One

Suggested online book for navigation in Immunology:

Gene Mayer, Denis Hudrisier:   http://www.microbiologybook.org/book/immunol-sta.htm

13In Focus: Zika virus
Zika virus was first detected in Rhesus monkeys in Uganda (Zika forest), as an adjunct to results in yellow fever research (1947). Similar to other pathogenic members of the Flaviviridae family (Dengue virus, West Nile virus, Japanese encephalitis virus, Yellow fever virus, Tick-borne encephalitis virus), the Zika virus (11kB single stranded (+)RNA in its genome)is transmitted by vectors (Aedes aegypti, Aedes albopictus mosquitoes bites) to the target organism. Opposite to other pathogenic members of the Flaviviridae family, the symptoms of Zika virus infection in humans are mild (skin rash, joint complaint, fever), and nearly 80% of those infected  were asymptomatic (so far). The latter may explain why no epidemic but sporadic cases of infection were detected until 2007. The first extensive epidemic was detected on Pacific Yap Island (Micronesia) in 2007, reflecting the escape of the virus from its endemic regions in Africa-Asia. According to the latest biogeographic information, the spread of Zika infection could be detected in Mexico, in Central America, the Caribbeans and also in Brazil [http://www.nejm.org/doi/full/10.1056/NEJMra1602113?query=featured_zika]
Further healthcare and social problems in Zika infections are the neurological abnormalities (microcephalia, Guillain-Barré syndrome) often associated with the virus infection [http://www.nejm.org/doi/full/10.1056/NEJMoa1601824?query=featured_zika#t=articleTop].
Worth to consider that biogeographical/regional discrepancies were observed in the severity of symptoms of Zika infection (e.g. Thailand with mild, Brazil with serious symptoms in infected patients). [http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(16)30010-X/fulltext]
Neither vaccines nor former research results were at hand up to now for immediate measures to take in prevention and localization of Zika infections.
In the latest WHO prognosis on the geographical spread of the virus, approximately four million new infections in the year 2016 are indicated. The sudden and fast spread of the virus in our days (i.e. infected passengers from wet, humid, warm climate → through vector mosquitoes → bites in recipients) and the highly populated subtropical areas, they all urge the availability of adequate medicines, mostly vaccines as required. However, the introduction of a new vaccine, the manufacturing process itself is very expensive, approaching billions of dollar in magnitude. Still, it is the efficacy-safety tests that take most of the costs.

February, 2016
Inovio Pharmaceuticals Inc. announced the invention of a candidate for synthetic DNA vaccine against Zika infection. In its preclinical phase of development, the product provoked strong antibody response and Tkiller cellular response in experimental mice. Human Phase I clinical studies are forecasted to start with this candidate vaccine by the end of the year 2016 [http://ir.inovio.com/news-and-media/news/].

March-April, 2016

Cryo-electron microcopic studies performed on Zika virus were published [Sirohi D. et al, 
Studies were run on virus strain
(H/PF/2013) isolated from a patient suffering from Zika infection in the epidemic of 2013-2014, in French Polynesia. When characterizing this strain, the coding region of the virus genome showed more than 99% nucleotide homology to that of the strain circulating this time in Latin America.
In the cryo-electron microscopic comparison of the icosaeder mature virions 
similarity was shown in the structural assembly of Zika virus, of Dengue virus, and of West Nile virus. Nevertheless, significant difference was found in the glycosylation of the Zika virus envelope protein ’E’ (a protrusion), a feature influencing spatial conformation of the protein. This acts as a causal factor in binding of the virus to target cell surfaces in the pathogenesis of infection. 
With regard to aspects of virology, immunogenicity or pathogenesis, the results published give the opportunity to compare Zika virus to other members of the Flaviviridae family; a step inevitable towards vaccine production. Further, the diverse data obtained may serve for the elaboration of a diagnostic standard, missing at present from everyday practice. [http://www.nejm.org/doi/full/10.1056/NEJMp1603734]

An important detail to consider for  diagnostic standard mentioned above, is the serological cross-reactivity of antibodies developed to Zika virus with antibodies developed to other members of the Flaviviridae family (e.g. Dengue fever virus, yellow fever virus), hampering thus the serological specificity of the diagnosis. Therefore, real time RT-PCR proved to be reliable in virus diagnostics when analysing blood plasma/serum as well as urine samples, according to the Brazilian practice of our days. Due to the earlier and shorter time window when analysing blood plasma/serum for the presence of viral RNA, it is the urine sample that bears the diagnostic advantage of longer time window with higher (better to evaluate) RNA level after onset of symptoms, and, opposite to blood taking, urine collection is a less invasive way of sampling.

A milestone on the way understanding Zika infection is the development of immunocompromised mouse models (lack of receptors for IFN α/β, little-if-at-all production of IFN α/β). In these experimental animals neurodegenerative alterations following Zika infection were accompanied by viral loads in the brain and the spinal cord, and the highest viral load was found in the testes. Since these experimental findings confirm data of human epidemics, the immunocompromised animal models together with their immunocompetent counterparts, are promising models for the follow up of the pathogenesis of Zika infection and, for the control of potential medicines and vaccines in preclinical phases of their development. 
[Lazear H.M. et al. (2016): A Mouse Model of Zika Virus Pathogenesis   Cell Host & Microbe 19, 1–11]
[Dowall S.D. et al. (2016): A Susceptible Mouse Model for Zika Virus Infection  PLoS Negl Trop Dis 10(5): e0004658. doi:10.1371/journal.pntd.0004658]


May, 2016
A WHO report summarizes risk assessment studies on Zika transmission in Europe with the calculated likelihood for local spread at country level [Zika Virus Technical Report; Interim Risk Assessment WHO European Region May 2016]. The assessment report is based on questionnaires answered by 51 states (+ Principality Liechtenstein) of the 53 targeted. Authors of the report when considering data obtained in questionnaires, classified the countries under study into categories according to the local likelihood of Zika transmission (high likelihood/moderate likelihood/low likelihood/very low likelihood/no likelihood), and the results were summed up graphically, too. 
Besides risk assessment, the WHO report also gives recommendations according to the level of likelihood for Zika transmission. The recommendations comprise of strategies with the four pillars of preventive and emergency care (integrated vector management; surveillance; laboratory capacity; emergency risk communication) included. According to the frequency of virus vectors Aedes aegypti and Aedes albopictus observed, for the action plans in recommendations, the authors of the report combined the categories of virus transmission likelihood resulting in two final categories with countries assigned to them (high + moderate likelihood// low + very low + no likelihood).

Restricting the spread of insects, vehicles for virus transmission ( vector control) is unavoidable in preventing and localizing infections. As for the way of managing it, let's have a look at the experiences collected by other continents

1. In their publication at the turn of this century, researchers from Cuba and Venezuela [Rodriguez Coto M.M. et al.(2000): Malathion Resistance in Aedes aegypty and Culex quinquefasciatus After its Use in Aedes aegypty Control Programs  J. Amer. Mosquito Control Association, 16: 324-330.] reported on the stable resistance to chlorinated hydrocarbons (like the fat soluble, carcinogenic, antiandrogenic and teratogenic dichlorodiphenyltrichloroethane-DDT, with characteristics of increasing ion permeability across membranes) in two species of insects; in Aedes aegypti and in Culex quinquefasciatus. After DDT, the next period of insecticides favoured the use of neurotoxic organic phosphates (OPs) targeting enzyme acethylcholinesterase. Among OPs it is temephos (acting on insect larva ≡ larvicide), phenitrothion and malathion (acting on adult insect ≡ adulticide) that had widespread use in practice. It was of great surprise that in spite of the intense and robust spraying of OPs in the Caribbean area in the preceding 15 years, the resistance to malathion of the Aedes aegypti populations remained at a low or slightly moderate level. Therefore, in restraining Dengue fever the vector control further relied on the use of malathion. Opposite to Aedes aegypti, populations of Culex quinquefasciatus developed strong resistance to malathion. This resistance is explained by the upregulation of insect genes coding for detoxifying esterases, more precisely, by the appearance of two esterase isoenzymes in Cuba, Venezuela, Columbia, Brazil, with different migration patterns in acrylamide gel electrophoresis. Resistance probably introduced by migration or passive dispersal was neither confirmed nor excluded. No data were found of the resistance developed to insecticides similar to natural pyrethrins, the Pyrethroids acting on voltage-gated Na+ channels in nerve cell axon membrane. 

2. A Brazilian-French research is reported in a publication of 2007 [da Costa-Ribeiro M.C.V. et al.(2007): Low Gene Flow of Aedes aegypti between Dengue-Endemic and Dengue-Free Areas in Southeastern and Southern Brazil    Am. J. Trop. Med. Hyg. 77: 303–309.], comparing the southeast (Dengue fever is endemic) region to the southern (Dengue fever free) region in Brazil. For the population genetic follow up of passive gene transfer, populations of Aedes aegypti were collected in 11 and 1 towns of the regions respectively. Samplings were performed in highly populated localities  minimum 1,2 km and  maximum 1,538.6 km in distance from one another, and, they all have ground transport connections to Rio de Janeiro. The sampling period lapsed from March 2003 - December 2003, in three-month intervals, with regard to rainy and dry seasons. The insect samples were cared for under standard circumstances (temperature, humidity, parts of day...) until reaching the adult stage in development. For biomarkers in the samples, isoenzyme (> genetic) polymorphism was chosen covering the same 10 enzymes per each of the samples (among them glucose-phosphate dehydrogenases, hexokinases, glycerophosphate dehydrogenases...). For isoenzyme polymorphism the reference strain Paea/Tahiti (French Polynesia/1994) was used in the electrophoretic pattern analysis. The evaluation of degree in differentiation, the indication of genetic variability in populations, was made according to the Hardy-Weinberg principle. It was determined, that Aedes aegypti populations under study were highly differentiated with changing patterns according to the rainy and the dry seasons, further, depending on transitions in weather periods and more, on habitat. Since genetic variability was found also in samples collected nearby the ground transport connections (autoroutes), the conclusion was drawn that regions of Brazil under study could not give rise to passive transmission of Dengue. Transmission of Dengue is performed by insect populations sensitive for the virus, that is they become carriers, however, according to theories, there is no opportunity for virus carrier state in insect strains of high and changeable genetic variability [Gooding R.H.(1996): Genetic Variation in Arthropod Vectors of Disease-Causing Organisms: Obstacles and Opportunities     Clinical Microbiology Reviews 9: 301–320.].

3. A report on the resistance status of Aedes albopictus collected in some areas of the United States was published in 2014 [Marcombe S. et al.(2014): Insecticide Resistance Status of United States Populations of Aedes albopictus and Mechanisms Involved   PLoS ONE 9(7): e101992. doi:10.1371/journal.pone.0101992]. Insect populations collected on the East Coast (five counties in New Jersey, one county in Pennsylvania, two counties in Florida) were studied in F1 progeny populations (eggs-larvae-pupae-adults) for the effect of 11 insecticidal compounds (6 of them larvicids and 5 of them adulticids) by monitoring two main mechanisms of developing resistance; target mutations and the increased expression of detoxifying enzymes. Results were referred to those  observed in an insect population (ATM95) sensitive to all the chemicals applied. It was demonstrated that larval populations of all the eight sampling sources had no strong resistance to the insecticides studied. Temephos (group OP of insecticides) proved to be effective in every larval populations of all sampling sources. Nevertheless, worth to consider the strong resistance to malathion (group OP of insecticides) in adult populations from Florida and, adult populations from New Jersey were already on the way to reach this kind of resistance

All the insect populations were sensitive to Pyrethroids. Concurrently, DDT resistance could be demonstrated in samples from Florida. This latter suggests the feasibility of developing cross resistance to Pyrethroids too, in Florida region, since dispersal of Aedes albopictus in the USA was preceded by the ban of DDT use (1972), further, targets for Pyrethroid action are in overlap with targets for DDT action (> voltage-gated ion channels). So, resistance to DDT of Aedes albopictus throwed shadow on the promising effects of Pyrethroids. Anyway, resistance to DDT in Florida region could be a consequence of passive transfer of Aedes albopictus populations already resistant to DDT, from Asia.

It is almost certain that the success of Aedes albopictus in its 'world tour' was due to passive transfer in the last 30-40 years, resulting the spread of this insect to all the continents, except Antarctica. In Africa (Cape Town), the first time of finding living larvae was in 1989, in used tyres shipped from Japan. Though the shipment was disinfected, still, two years later the insect was also detected in Nigeria, Cameroon, Equatorial-Guinea and Gabon. The presence of Aedes albopictus was indicated In Central and Latin America from the 1980s-1990s; the established populations of Mexico and Brazil began to colonize other countries too, in a southern and eastern direction, by expansion. By the end of the 18th century, the insect was identified in area Hawaii; the continental US appearance was registered in 1985, in Texas. From that time on, the presence of the insect was indicated in sequence, in 866 counties of 26 states. In Europe, it was Albania (1979) where the first signal of this insect was given. Next stations in spread were the Mediterraneum, and the countries of the near East. From here, the populations of Aedes albopictus reached regions of the Netherlands, Switzerland, France, Belgium and Germany too, in northern direction and delayed expansion. 
It seems that the winner of the competition between Aedes albopictus and  Aedes aegypti  is the former, with the ability of the eggs to keep diapause (dormancy) in development as a response to environmental situation less advantageous (in temperature, in humidity, in precipitation...). If environmental situation turns back to normal, the eggs go on in their developmental program. At gene level, the dormancy period is supported by the transcription profiles of two genes (Ae. albopictus Pepck, PCNA); are they promising targets for vector control at gene level?    
[Bonizzoni M. et al.(2013): The invasive mosquito species Aedes albopictus: current knowledge and future perspectives    Trends Parasitol.  29: 460–468. doi: 10.1016/j.pt.2013.07.003]
The drawbacks of using chemical insecticides (issues of > selectivity often missing in targeting > environmental damage caused > incorporation into food chain > resistance developed) gave boost to look for new ways in vector control, to restrict and limit the growth of insect populations carrying infective viruses. 

4. Take a look at some options provided by research in physics, biology, and mathematics.

  • SIT (sterile insect technique): sterility of insect males induced by ionizing radiation in laboratory circumstances [mutations provoked in reproductive cells too, at chromosomal and gene levels]; an open field release and a later recapture of these sterile male populations make possible to follow their behaviour in natural habitats. Advantages  of the method: environmentally friendly, selective, no opportunity for the next generation. Disadvantages of the method: due to technical difficulties in application > density dependent reactions in insect populations > uneven absorbed dose > physical status of irradiated males restrict their capabilities to compete with counterpart wild males in preventing wild mating.  

  • RIDL (release of insects carrying a dominant lethal), a biotechnological version of SIT. A lethal transgene construct with a dominant (developmental) trait repressed in laboratory circumstances, integrated into insects' genome. Open field release of these insects results derepression of the repressed dominant trait, that is given to progeny generations when mating in natural habitats. Thus, the derepressed dominant trait serves for preventing the offspring to reach its mature adult form unwished.  
    • The AeAct-4 (actin) gene of the mosquito Aedes aegypti is responsible for the regulation of wing muscles movements; the expression of the gene is enhanced in females (the so-called "buzzy oscillation" attracting males). After integration into larval genome of a transgene contruct with AeAct-4 (actin) gene promoter + intron sequences in it, the developing females showed failure in wing movements. This is a lethal trait, since females with no wing movements are not capable to escape from danger or to find food any more [Guoliang Fu et al.(2010): Female-specific flightless phenotype for mosquito control    Proc.Natl.Acad.Sci.USA. 107: 4550-4554.]
    • The method above was elaborated for mosquito Aedes albopictus, too. After integration into larval genome of a dominant lethal transgene construct with AeAlbAct-4 gene promoter sequences in it, the developing females were characterized by failure in wing movements. It was concluded, that promoter sequences of Act-4 (actin) genes regulating wing movements in the two mosquito species, are interchangeable [Labbé G.M.C. et al.(2012): Female-Specific Flightless (fsRIDL) Phenotype for Control of Aedes albopictus  PLoS Negl Trop Dis 6(7): e1724. doi:10.1371/journal.pntd.0001724]    
    • Open field dispersal study on the competitive fitness in mating of genetically modified insect males: the OX513A-My1 sterile male population of Aedes aegypti carrying a dominant lethal transgene construct with fluorescent marker included, was  released with wild counterparts in a forest area with no inhabitants in Malaysia, on december 21, 2010 daytime, at temperature 32,6 oC, relative humidity 66%, air movement 1m/s, without precipitation. Recapture was made in the period of december 22, 2010 to january 5, 2011, at a rate of 50% and 17% of mutant and wild populations, respectively. Results: the estimated maximum distance after release was 250 m; the average distance in mutant populations was slightly less // the estimated average lifespan was 2 days (mutant) and 2,2 days (wild), meaning, that due to transgene construct, no significant alteration of this parameter was observed. Repeats of dispersal experiments are planned in urban environment with female mosquitoes' habitats. Main purpose is the reduction of vector populations carrying infective agents > dominant lethal alleles are placed in the progeny by sterile males at mating, therefore, the offspring does not reach its mature adult form.
    • Dispersal experiment similar to above, performed in suburb of Juazeiro, Brazil, and published in 2015 [Winskill P. et al.(2015): Dispersal of Engineered Male Aedes aegypti Mosquitoes   PLoS Negl Trop Dis 9(11): e0004156. doi:10.1371/journal.pntd.0004156]. Using multivariate analysis, the authors  searched for optimum description of mutant dispersal. 

  • Gene silencing by double stranded/dsRNA sequences induced small interfering RNA/siRNAs. As for the unpredictable evolutionary consequences of transgene solutions, the regulatory insufficiencies in this field, the public aversion towards it, they all, opened door to new biological approaches in vector control. In 2 days old Aedes aegypti the testis specific genes determining male character (10 genes) and the gene determining female character were identified by subtractive hybridization. Total RNAs of pupal and mature developmental forms were isolated for obtaining dsRNA sequences targeting the previously identified specific genes determining the male and female characters. Larvae (with bacterial vehicle) and pupae (by injection) were fed with the target sequence specific dsRNAs obtained. The dsRNAs induced siRNA functions (transcriptional/post-transcriptional shutdown) on target gene sequences ended up in the stable manifestation of sterile male and inhibited female (no sex) character. Advantages of the method: no need for irradiation, for transgene constructs, for selection of males or females. The method is based on molecular protective classic mechanisms of eukaryotes, mechanisms specific for target sequences so as the vital functions of the resulting insects with no sex determination are retained, expectedly leading to competitive fitness in mating. The method is transferable to large scale production and for other species in vector control. [Whyard S. et al. (2015): Silencing the buzz: a new approach to population suppression of mosquitoes by feeding larvae double-stranded RNAs  Parasit Vectors. 2015; 8: 96.  doi: 10.1186/s13071-015-0716-6.]      
  • RNA guided CRISPR/Cas9 mediated genome editing (type II): an adaptive protecting mechanism in prokaryotes leading to cleavage, heritable editing of foreign nucleotide sequences (plasmid, virus) integrated in the prokaryote genome. Participants of the mechanism are the following: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas9 (CRISPR associated 9 ≡ endonuclease cleaving dsDNA). Principle of the method: transcription of CRISPR interspaced sequences and adjacent short palindromic sequences into total RNA > formation of transactivating tracrRNA from total RNA > with the help of tracrRNA formation of crRNA complementary to interspace sequences from total RNA > hybridization of tracrRNA to complementary palindromic sequences > activation of RNase III enzyme > tracrRNA and crRNA liberated, form association complex with enzyme Cas9 > complementary to crRNA interspaced dsDNA is the target for enzymatic cleavage by complex Cas9 > dsDNA breaks are repaired by DNA repair machinery using nonhomologous end joining. Result: deletion, insertion (> mutation) and a disrupted target gene. Advantages of the method: the result of cleavages on integrated plasmid /virus sequences is inherited to offspring ('genetic memory'). According to experimental results so far, the plasmid construct CRISPR/Cas9 prepared for Aedes aegypti needs further development. [Dong S. et al.(2015): Heritable CRISPR/Cas9-Mediated Genome Editing in the Yellow Fever Mosquito, Aedes aegypti     PLoS One. 2015; 10(3): e0122353.doi: 10.1371/journal.pone.0122353] 
  • Biocontrol with cytoplasmic endosymbiont bacteria Wolbachia pipientis.   The supporting evidence as below.                                                                         
1. Arbovirus (arthropode borne virus) spread depends on the interactions among the virus, the insect vector and  several microbial components. According to observations, Drosophila C (RNA) virus infection fatal to Wolbachia-free populations of Drosophila melanogaster (fruit fly), results in delay or decrease of insect deaths, and also of virus accumulation in fruit flies with Wolbachia endosymbionts. Restricted to RNA viruses, the Wolbachia protection against viruses is based on its high cytoplasmic density both in somatic and reproductive cells. A competition among the parasite virus, the cytoplasmic endosymbiont bacterium and the vector/host cell (of Drosophila, of mosquito), in reaching nutrition and energy source is presumed to manifest, giving evolutionary advantage to Wolbachia of high density. The mechanism of Wolbachia protection against (RNA) viruses is related to generation of reactive oxygen intermediates (ROI), to enhanced activity of regulatory miRNAs (in gene silencing), and to the immune response provoked in the vector organism. [Johnson K.N. (2015): The Impact of Wolbachia on Virus Infection in Mosquitoes   Viruses 7: 5705–5717.   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4664976/]  
2.  It was in 1924 when Wolbachia was first demonstrated in mosquitoes (Culex pipens); however, for different microbiomes in different mosquito genera, Wolbachia could not inhabit all of them. The introduction of Wolbachia in biocontrol of insect vectors carrying pathogenic viruses is based on the endosymbiontic capability to manipulate the reproduction of the host organism by prioritizing its own bacterial reproduction. This leads to deformities induced in the host: to parthenogenesis, to feminization (> cytoplasmic thrive in vertical transmission), to killing of males (> failures in DNA packaging of male reproductive cells), to cytoplasmic incompatibility (offspring free of Wolbachia goes through deleterious embryonic development). 
Among others, mosquitoes Aedes aegypti and  Aedes albopictus were 'transinfected' transiently and stably as well, with Wolbachia strains of diverse activity, of Drosophila origin. Compared to wild populations, the behaviour pattern and the "fitness" (competitive mating) of the 'transinfected' insect populations were studied. The conclusions denote the differences in results obtained from 'transinfections' with different strains of Wolbachia.  [Jeffries C.L., Walker T. (2016): Wolbachia Biocontrol Strategies for Arboviral Diseases and the Potential Influence of Resident Wolbachia Strains in Mosquitoes   Curr Trop Med Rep. 2016; 3: 20–25.  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4757633/   
Emerging questions: 
1. At an evolutionary scale, what kind of maneuvres will be deployed by members of the triad: the pathogenic virus - the endosymbiont Wolbachia - the insect vector,  to surpass 'transinfection' pressure?   
2. Spread of the endosymbiont Wolbachia is realized in horizontal (somatic) and vertical (reproductive) transfer, too. Regarding 'transinfected' hosts, what are the cargo and, how and where are they taken by Wolbachia in spread?

G-15G-15 G-15 

June, 2016
A report was published on the preventive effect of interferon induced transmembrane proteins (IFITM) in early phase of Zika infection in cultures of HeLa, Vero, and A549 cells stably transduced with a lentivirus-IFITM construct
[Savidis G. et al.(2016/): The IFITMs Inhibit Zika Virus Replication  DOI: http://dx.doi.org/10.1016/j.celrep.2016.05.074]. According to the report, it was IFITM1 and IFITM3 of the IFITM proteins restricting pathogenic viral infections that showed effects as expected. Based on the cellular level of virus-RNA measured, IFITM3 proved to be stronger in preventing early phase of virus infection. As for the mechanism of prevention, authors of the report suggest a direct action of IFITM3 on cell membrane and/or virus membrane, interfering thus with membrane fusion and pore formation so that viral genome and accessory key enzymes cannot enter the host cell. Regarding that gene expression of IFITM proteins at basic level is the highest in case of IFITM3, a protection performed by IFITM3 in the early phase of Zika infection is presumed. Further, IFITM3 may also act as a spark in starting interferon induced chain reaction of gene expressions. Considered as components of the innate immunity, IFITM proteins are able to modulate significantly the early and decisive phase of pathogenic virus infection. Confirming studies are still needed (e.g. establishment of IFITM-deficient in vivo models).


June-July-August, 2016
Recent preclinical results in Zika vaccine development point at the efficacy of experimental plasmid DNA vaccine {Zika virus immunogen coding expression system (complete: virus pre-membrane + Env components, partial: only-Env component)}, and at the efficacy of inactivated Zika virus vaccine {conventional vaccine} in tests performed in mice and in Rhesus monkeys models.
Zika source for the animal tests were: virus isolates of the Asian ZIKV16 line (ZIKV-BR/Brazil ZKV2015 >  BeH815744  and  ZIKV-PR/Puerto Rico > PRVABC59), differing in 5 aminoacids

Mouse Model
Immunogenic studies: Balb/c  mice (5-10/group) immunized once (50 μg DNA vaccine/i.m.). For detection of specific antibodies ELISA tests were performed on week 3 after immunization.  
  • Compared to the only-Env-DNA vaccine, the complete (pre-membrane + Env) DNA vaccine provoked higher titers in Env specific antibodies. No antibody response specific to the pre-membrane component (alone) was demonstrated. 
  • Zika virus specific neutralizing antibodies were generated by the complete (pre-membrane + Env) DNA vaccine (microneutralizing test). 
  • Env-specific TCD4+  and  TCD8+ lymphocyte response was induced by the complete (pre-membrane + Env) DNA vaccine (IFN-γ ELISPOT test + multiparametric intracellular cytokine staining). 
Evidence for the protective effect of DNA vaccine: virus challenge to Balb/c mice after immunization (week 4) and to control mice {i.v. ZIKV-BR  or  ZIKV-PR / 105 virus (102 PFU)}. Quantity of viral load was controlled by RT-PCR. 
  • In control animals viremia was established in 6 days. 
  • ZIKV-BR viremia was prevented (virus number < 100 copies/ml) at different test times (n = 10) by complete (pre-membrane + Env) DNA vaccine. Similarly, complete protection was observed following virus challenge on week 8 after immunization. Protection was also conferred by complete (pre-membrane + Env) DNA vaccine when virus challenge with ZIKV-PR strain was performed. 
 Immunogenic studies: Balb/c  mice (5/group) immunized once  with  inactivated Zika virus vaccine (1 μg / i.m. or s.c., strain Puerto Rico PRVABC59).  For detection of generated antibodies, ELISA tests were performed.
  • Compared to inactivated Zika virus vaccine/s.c.the inactivated Zika virus vaccine/i.m.  resulted higher antibody titers in the immunized animals. Both types of delivery succeeded in generating Zika virus specific neutralizing antibodies
  • Parenteral (i.v.) virus challenge (ZIKV-BR strain) on week 4 after immunization; complete protection was achieved in animals immunized by the intramuscular route.  
Adoptive transfer: IVIG (intravenous immunoglobulin)
Given to recipients, IgG purified from sera of Balb/c mice immunized with complete (pre-membrane + Env) DNA vaccine (ZIKV-BR) provided antiviral protection at a strength proportional to the Env specific antibody titer of the immunized donor. In other words: antiviral protection is conferred by IgG on condition the Env specific antibody titer exceeds a critical threshold level.
[Larocca R.A., Abbink P. (+24) (2016): Vaccine protection against Zika virus from Brazil     Nature Letter Accelerated Article Preview published online 28 June 2016]

Rhesus Model
 Immunogenic studies: inactivated Zika virus vaccine (s.c.) to 16 Rhesus monkeys (5 μg/animal) on week 0 and 4.  The generated antibodies were checked by ELISA method. 
  • Two weeks after the first immunization the rise of Env specific antibodies and Zika virus specific neutralizing antibodies (mikroneutralizációs teszt) were detected in the animals. Following the second immunization (week 4), on week 6, the antibody titer got even higher. Compared to control animals, in majority of the immunized monkeys, Env specific moderate cellular immune response was elicited (IFN-γ ELISPOT test).
Evidence for the protective effect of inactivated Zika virus vaccine: virus challenge {ZIKV-BR or ZIKV-PR / 106 virus (103 PFU)} to immunized and control Rhesus monkeys (s.c.). Quantity of viral load in host animals was detected by RT-PCR, virus infectivity was confirmed in Vero cell culture.
  • In control monkeys viremia was established within 6-7 days after virus challenge. Virus was detected in urine, in cerebrospinal fluid (CSF) on day 3; in colorectal and cervicovaginal secretions on day 7. No significant difference was shown in plasma viral load following ZIKV-BR or ZIKV-PR challenge.  
  • Complete protection of monkeys against Zika infection was provided by inactivated Zika virus vaccine (virus number in blood /urine /CSF/colorectal secretions /cervicovaginal secretions  < 100 copies/ml)
Adoptive transfer: IVIG
  • Plasma IgG was purified (protein G affinity chromatography) from monkeys immunized with inactivated Zika virus vaccine (week 8 after immunization)Balb/c mice were infused with the IgG obtained. Virus challenge {ZIKV-BR /105 virus (102 PFU)} to mice following infusion: proportional to IgG dose infused, protection against Zika (high dose) and decreased viremia (low dose) were observed. 
  • Naďve Rhesus monkeys were infused with IgG as above. Following virus challenge {ZIKV-BR/106 virus (103 PFU)}, one of the recipients given high dose IgG showed complete protection against Zika, while the other of the recipients showed  slight viremia on days 3-5. Virus replication was not supported by IgG in subtherapeutic doses. 
In brief: according to teaching of experiments in rodents and primates, adoptive transfer of IgG from donors immunized with inactivated Zika virus vaccine is promising tool for conferring protection against Zika.
[Abbink P., Larocca R.A. (+ 36) (2016): Protective efficacy of multiple vaccine platforms against Zika virus challenge in rhesus monkeys   Science  04 Aug 2016:]  

66 6

September, 2016

WHO Situation Report comprising data up to 14th September, notifies about the so far revealed two lineages of Zika virus, the lineage Zika-Africa and the lineage Zika-Asia.
  • Lineage Africa is observed in Africa; seven new registered infections were characterized by nucleic acid sequencing, in Guinea-Bissau.
  • Strains of lineage Asia are documented in Asia, in Western Pacific Region, and in the Americas.
  • Neurological complications are related to after-2007-strains of Zika- Asia. However, considerations should be extended to any strains/lineages, due to shortages in current diagnostics and knowledge.
  • After-2007-strains of Zika-Asia were isolated in French Polynesia (since 2013), in the Americas (since 2015), in Cabo Verde (2016).
  • Since 2007 altogether 72 countries/territories have announced mosquito mediated Zika infection. 70 countries/territories of them gave the first infection signal in 2015.
  • Since February, 2016 we are aware of Zika infection transmitted by non-mosquito (human) vector (signalled in five countries of the Americas, in six countries of the European continent, and, in a country of the Western Pacific Region).
  • The potential relationship between congenital malformation of the central nervous system and Zika virus infection is underpinned by reports from 20 countries/territories.
According to Zika molecular evolution, the process has progressed by genetic recombinations resulting three distinct genotypes so far; the East-African cluster (cca. period 1892-1943), the West-African cluster (evolved in several streams of cca. 1935-1940; cca. 1950-1960; cca. 1965-1985, cca. 1995), and the Asian cluster (cca. 1960). As for phylogenetic analyses, the spread of virus from East Africa to direction West Africa and Asia, then to Western Pacific Region and the Americas, is presumed.

Early in 2015 consideration: genome of Zika virus settled in North-Brazil is comparable with the genome of Zika virus isolated in French Polynesia, in 2013. This latter belongs to the Asian cluster. 

consideration: nucleic acid sequencing
(NGS = next generation sequencing) of Zika virus settled in Guatemala and Puerto Rico, further, their evolutionary estimation (GenBank KU501215/Puerto Rico PRV-ABC59; GenBank KU501216/Guatemala 8375; GenBank KU501217/Guatemala 103344) support the notion that Zika genomes of Guatemala and Puerto Rico strains are similar to Zika genomes of North-Brazil and French Polynesia strains; they all belong to the Asian cluster.

Nucleic acid similarity among Zika virus genomes isolated in the western hemisphere, are >99%. 

Nucleic acid similarity among Zika virus genomes isolated in the western hemisphere, in East Africa, in West Africa, show about 89% similarity.  


October-November, 2016

WHO Situation Report, 13 October 2016  (for comparison see WHO Situation Report, 15 September 2016)
  • Since 2007 altogether 73 countries/territories have announced mosquito-mediated Zika infection.   
In September the number was 72 countries/terrritories.

  • 67 countries/territories of the 73 mentioned gave the first signal of infection in 2015. 
→ In September the number was 70 countries/territories.

  • The potential relationship between congenital malformation of the central nervous system and Zika virus infection is suggested by reports from 22 countries/territories. 
→ In September the number was 20 countries/territories.
Related to the latter, to Zika neurotropism, the potential far reaching consequences of it provoked debates unfolding significant diversities in professional standpoints.
Two examples of them are shown below.

Summarizing thoughts/1
[Jing Wu et al. (2016): Available Evidence of Association between Zika Virus and Microcephaly  Chin Med J (Eng). 129: 2347-2356.]   
As for international publications registered in PubMed in time period running from the first detection of Zika virus in humans in Niger 1950, till 27 May 2016, the authors are not convinced about the causal relationship presumed between Zika virus infection and multifactorial congenital microcephaly.
Questions raised, links missed
?  thorough tracking of pregnancies exposed to Zika virus; detailed laboratory tests for detection of virus and of       immune analytes; use of ultrasound examinations ?
?  economic environment and conditions of public nutrition in countries involved in virus epidemic ?
?  interference of Zika with other virus-, bacterial, parasite infections ?
?  standards in microcephaly diagnostics ?
?  virus mutations resulting the accelerated epidemic of autumn 2015, expanded later throughout the continent       of Latin America ?
?  mechanisms explaining Zika neurotropism ?

Summarizing thoughts/2
[Jin-Na Wang and Feng Ling (2016): Zika Virus Infection and Microcephaly: Evidence for a Causal Link   Int J Environ Res Public Health. 13: 1031-1054.]
Opposite to Summarizing thoughts/1 while evaluating international studies registered in PubMed, the authors here are less reserved in adopting the causal relationship presumed between Zika virus infection and multifactorial congenital microcephaly. The preliminary report on the ongoing studies in Brazil is taken as supporting rationale.
As for the authors, the causal relationship is evidenced by detecting virus particles in placental chorionic villi and in brain tissues. However, Jing Wu et al. pointed on the lack of laboratory tests periodically performed on blood and urine samples. Similarly, on basis of studies published so far, the pathogenesis of Zika microcephaly is classified unresolved as for Jing Wu et al (Summary/1). Dissenting from the opinion of Jin-Na Wang et Feng Ling, Jing Wu et al. consider the experiments on mice not comparable to human cases.

Both summarizing thoughts urge the involvement of non-human primates in further experiments.

Steps ahead to resolve disagreements as above:

First prospective case-control study (Brazil, 15 January - 2 May 2016), preliminary report:
[de Araújo TVB (+19): Association between Zika virus infection and microcephaly in Brazil, January to May, 2016: preliminary report of a case-control study  
Studies were run in eight public hospitals (Recife, Brazília) on 32 newborns with microcephaly and 62 healthy newborns (controls chosen by the parameters of: same living areas, expectedly same date of birth, without microcephaly). Among the 32 newborns with microcephaly, in 13 (41%) babies the Zika infection was confirmed by laboratory tests. No Zika infection was detected in control babies.
Analyses of sera and cerebrospinal fluid samples for detecting Zika specific IgM and detecting Zika virus by serology and quantitative RT-PCR, respectively. Analyses of maternal sera by Zika and Dengue virus specific plaque reduction neutralizing test (PRNT).

In progress:
prospective cohort studies in support of the presumed causal relationship between Zika infection and developing congenital microcephaly.
Preliminary reports on the ongoing studies are found here: 
[Brasil P, Pereira JP, Jr, Raja Gabaglia C, Damasceno L, Wakimoto M, Ribeiro Nogueira RM, et al.(+14): Zika virus infection in pregnant women in Rio de Janeiro – Preliminary report. N Engl J Med. 2016 [Epub ahead of print]. doi: 10.1056/NEJMoa1602412.]
[Villamil-Gómez WE, Mendoza-Guete A, Villalobos E, González-Arismendy E, Uribe-García AM, Castellanos JE, et al. Diagnosis, management and follow-up of pregnant women with Zika virus infection: A preliminary report of the ZIKERNCOL cohort study on Sincelejo, Colombia. Travel Med Infect Dis. 2016;14:155–8. doi: 10.1016/j.tmaid.2016.02.004.]

November 2016: recruiting for Phase 1 clinical trial No. NCT02963909
First Zika vaccine candidate in human clinical trial: alum-adjuvated Zika virus Purified Inactivated Vaccine (ZPIV) in Phase 1 clinical trial.
Randomized, double-blinded, placebo-controlled trial for testing prevention, safety, efficacy, immunogenicity  in healthy  (Flavivirus-naďf and Flavivirus-primed), aged 18-49 volunteers of both genders. Estimated date of final data collection is May, 2018.        
(Source: ClinicalTrials.gov)


November-December, 2016

Zika - Dengue cross-neutralizing antibodies in preventing Zika infection
X-ray crystallographic and cryo-electronmicroscopic studies evidenced the dynamic changes in the conformation of Zika and Dengue surface 'E(envelope) glycoproteins in early phase of virus infection. These dynamic changes manifest in the course of specific interactions between virus and target cell surfaces (virus adhesion, virus docking), then in the course of membrane fusion carried about by virus surface and target cell (or cell compartment) membranes. The membrane fusion is a step critical and decisive in the process of virus infection. Regarding experimental data, membrane fusion mentioned above is inhibited by Zika-Dengue cross-neutralizing antibodies isolated from patient with Dengue infection, having "locking effect" on actual conformation of virus 'E' glycoproteins ("super serogroup") in surface topography. The results so far may give support to simultaneous prevention of Dengue and Zika infections (vaccine development?) as well as to a reliably broadened diagnostic window (serodiagnostic test development?). 
Evidence 1
Neutralizing all four Dengue serotypes, broadly neutralizing human antibody to Dengue virus (to envelope 'E' glycoprotein dimer epitopes -EDE-) is also efficient in neutralizing Zika infection in vitro, in Vero cell cultures. 
[Barba-Spaeth G. et al. (+15): Structural basis of potent Zika–dengue virus antibody cross-neutralization  Nature 536, 48–53  (2016)  doi:10.1038/nature18938]

Evidence 2
H and L chain variable region sequences of human antibodies to Dengue virus cloned into plasmids > plasmids for transient transfection of human HEK293T cells  > purification of monoclonal soluble antibody C10 produced > preparation of IgG-Fab (antigen binding) fragments > in vitro reaction of IgG-Fab fragments with Zika virus (pH=8.0; pH= 6.5; pH=5.0) >>> cryo-electronmicroscopy of the interaction between 'E' glycoprotein dimer(s) + Fab(s).
[Zhang S. et al. (+10): Neutralization mechanism of a highly potent antibody against Zika virus  Nature Communications 7, Article number: 13679 (2016) doi:10.1038/ncomms13679]


13In Focus: HIV/AIDS virus

The HIV (human immunodeficiency virus) is an icosaeder enveloped virus (family Retroviridae / genus Lentivirus / 9749 nucleotides - two copies of single (+)RNA strands in the genome) with surface spikes composed of glycoprotein (gp) trimers built up from gp120/gp41 dimers in special spatial arrangement. The binding of the virus to the target cell is mediated by these surface glikoprotein spikes. During productive infection it is the viral surface gp120/gp41 glycoproteins that bind to surface receptors and coreceptors (CD4 … chemokine receptor CCR5 … chemokine receptor CXCR4) on cellular elements of innate and adaptive immune responses (macrophages, dendritic cells, CD4+ T lymphocytes). After fusion of the viral membrane with the target cell membrane along with the activation of post-receptor signalization, the target cell is further restrained in its vital functions, ultimately leading to cell damage. The process is amplified by the multiplication (reverse transcription) of viruses accompanied by mutations in infected cells and, virions liberated from living and dead cells continue to infect other target cells, too. Finally, in majority of cases, after latency time, the resulting virus load ends up in fatal immunodeficiency of the host organism (Aquired Immunodeficiency Syndrome - AIDS).
An alternative to HIV is the SIV (simian immunodeficiency virus) targeting primates. The SIV infection, mainly in apes, leads to a condition similar to that of the HIV infection mentioned before, mostly in Africa. The virus strain SIVcpz detected in chimpanzee in 1999, was genetically almost identical to human HIV-1 virus and, the SIV virus detected in Cercopithecus (Old World monkeys) was genetically closely related to HIV-2 virus.
The virus strain SIVcpz is suggested to evolve from the recombination of two different SIV strains (after horizontal transfer). It is supposed that smaller monkeys infected with SIV strains different in their genomes were the prey animals for predator chimpanzee. Since the smaller prey animals were eaten, the predator chimp became a carrier of the different virus strains consumed with the preys. Consequently, the SIV strains carried by the predator were free to combine with one another in their new host and, after a further increase in virulence, they could cross the chimp-human barrier as well, in a prey-predator manner similar to that mentioned before. This last transmission may have happened around 1920, in Kinshasa - DR Congo. After transmission to the human body,
these SIV strains continued their propagation as HIV, in diverse lineages of HIV-1 virus (HIV-1 M,N,O,P) and, in subtypes (A,B,C,D,F,G,H,J,K) of the most frequent M lineage of HIV-1.
Even if different from HIV-1, the HIV-2 virus may have arrived in the human body in a similar "nutritive" way, however, in this case the Cercopithecus is supposed to serve as the prey animal. Since its first detection, the geographical spread of HIV-2 virus has been mainly localized in West Africa (Bissau-Guinea, Senegal). In the majority of cases, HIV-2 infections do not end in AIDS syndrome. If it happened at all, the overwhelming plurality of HIV-1 virions could be detected in the patient.
According to biogeographical data, it is the M lineage ’B’ subtype of HIV-1 virus to be detected most often in our days.
[In details > Paul M. Sharp and Beatrice H. Hahn(2011):  Origins of HIV and the AIDS Pandemic. Cold Spring Harb Perspect Med. Sep; 1(1): a006841  >http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3234451/   és  > http://www.pbs.org/wgbh/pages/frontline/aids/virus/tree.html]


According to data in the WHO UNAIDS booklet of 2015, the number of HIV infections registered in 2014  was 36.9 million. Among them 17.1 million were not aware of their infected status.  Although a global decrease in the number of infections could be seen, still, there were 2 million new infections and 1.2 million deaths of AIDS reported in the subject year 2014. [http://www.unaids.org/sites/default/files/media_asset/AIDS_by_the_numbers_2015_en.pdf]

Which was unbeleivable 15 years ago: approximately 15.8 million of HIV infected had access to antiretroviral therapy, according to the report from June 2015. Calculating with the access to antiretroviral therapy, the main goal declared by WHO to reach until 2030 is the global abolishment of the AIDS epidemic. Meanwhile this main program, the interim goal until 2020 is that 90% of the infected be aware of their infected status, 90% of the latter have access to antiretroviral therapy, and in 90% of the latter the antiretroviral therapy result a significant decrease in virus number. A further interim goal is that the current number of new infections decrease by 75%  and, the number of intrauterine and postnatal (breast feeding) infections (vertical transmissions) reach and remain at zero level.   
Calculating with the infected regions, and with emphasis on low income countries, the estimated cost of reaching interim goals by 2020  is  31.1 billion US $, a peak in costs expectedly reduced in later phases of the program.

The UNAIDS data reporting a decrease in the number of HIV infections highlight the availability of current antiretroviral therapy and suggest further development of adequate drugs targeting virus-cell interactions or critical steps in virus replication. Even so, the cardinal aim of our days in the fight against HIV/AIDS is the development of neutralizing antibodies against the HIV virus. These antibodies -vaccines- supplementing the actual antiretroviral therapy, could be a choice in the prenatal and postanatal care of infections, and mostly, in the prophylaxis of infections. This latter undoubtedly could generate a positive move in the economic indicators of the countries concerned. The greatest challenge in vaccine development is the highly varying character of the antigen determinants in the HIV virus envelope (increased virus growth rate accompanied by increased mutation rate resulting in great variety of viral surface epitopes).
In harmony with those above, interesting new approaches in vaccine research and development were published. [Summarized in > So Youn Shin (2016): Recent update in HIV vaccine development Clin.Exp.Vaccine Res. http://dx.doi.org/10.7774/cevr.2016.5.1.6.]

A remarkable approach is based on the well-known observation that in 20% of HIV infected patients, 2-3 years after infection, so called broadly neutralizing antibodies (=bNAbs) could be detected in the blood. These antibodies due to the 2-3 years of evolutionary selection (clonal selection) became capable to neutralize virus isolates harboring different surface antigens. Introduced into primates via passive transfer, the bNAbs restrained virus progression in infected animals, and prevented new infection in healthy ones. No data are available of the efficacy of bNAbs via passive tranfer, in humans. 
Taken together, the generation of bNAbs by active immunization became the goal to achieve. For the experimental production of bNAbs, the immunogen substance was assembled from different, artificially constructed HIV envelope protein components. It turned out, that the success in constructing and in immunizing with the HIV envelope protein components, depends on the binding of the first immunogenic component to the germ line B cells, on the activation of the rare precursor B cells potentially capable to develop into B cells producing bNAbs. In the publication the construction of  HIV immunogen vaccine candidates are presented [Joseph G. Jardine, Daniel W. Kulp,  Colin Havenar-Daughton (+19) (2016): HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen. Science:  351:  1458-1463].  

Another remarkable approach was published two years earlier. In its content a dynamic, other than usual way of thinking, a paradigm shift is disclosed.  
This research was based on the observation that HIV infection at body temperature (37 °C) is brought about by the interaction of virus and target cell surface glycoprotein epitopes
(gp120/gp41 … CD4 … CCr5/CXCR4 ternary complex), characterized by continuous and dynamic change in their spatial appearances (variations in spatial conformation, induced exposure or hiding, approaching to... or, move away from...), before membrane fusion (prefusion). This continuous and dynamic change in surface epitopes results in transient and short-lived prefusion surface patterns hence, the immune system has no time enough for recognition and for generation of specific antibodies in response (a possible cause of failure in the development of previous vaccines). Since the interaction and later the fusion, of virus surface and target cell membrane are temperature dependent processes, the temperature guided (<25°C) synchronous arrest of the prefusion surface pattern results in the fixation of the transient interactions of surface epitopes, preventing thus the fusion of membranes.
The considerations above led to the implementation of a coculture system, in which virus donor B cells were cocultured with virus acceptor T cells, in vitro.  After the prefusion synchronous arrest at different temperatures
(<25°C), the surface patterns of epitopes were fixed by 0,05% formaldehyde. Mice were immunized with this "designed antigens". The monoclonal antibodies produced by the animals did not  react with the "static", classic virus epitopes. According to expectations, the bNAbs produced by these "designed antigens" help to avoid virus-target cell membrane fusion, the step initiating productive HIV infection. 


This method of obtaining "designed antigen" may be beneficial for other viruses, too. A further advantage is its applicability in developing topical microbicides.         
[Tuckweng Kok, Adriana Gaeguta, John Finnie, Paul R Gorry, Melissa Churchill and Peng Li (2014): Designer antigens for elicitation of broadly neutralizing antibodies against HIV. Clinical & Translational Immunology  3, e24;  http://www.nature.com/cti/journal/v3/n9/full/cti201422a.html]

An alternative to prefusion synchronous arrest is the research published two years later (in 2016), describing the crosslinking (by glutaraldehyde, further, by EDC/NHS = [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysuccinimide]) of antigenic trimers (> surface and transmembrane glycoproteins) of virus surface envelope, to stabilize their conformational flexibility in a native-like form. The latter makes it possible for neutralizing antibodies (bNAbs) to access and, bind to corresponding antigens of the crosslinked complex. This binding is confirmed, and the heterogenous population of antibodies is separated into fractions, by affinity chromatography, wherein weak or non-neutralizing antibodies are not bound to antigenic complexes stabilized by crosslinking agents. The publication gives support to the idea that aiming at vaccine development, the stabilization of flexible surface antigenic determinants is inevitable for obtaining neutralizing antibodies (bNAbs) full of function. [Schiffner T. et al (2016): Chemical Cross-Linking Stabilizes Native-Like HIV-1 Envelope Glycoprotein Trimer Antigens J.Virol. 90: 813-828.   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702668/]  

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As long as there is no effective HIV vaccine...
  • Clinical efficacy of vaccine candidates: collected data (2013) ...

  • IFE-UNAIDS economic and ergonomic considerations for low and middle income countries from point of HIV vaccination ...
  • January, 2015 - January, 2017, South Africa, 6 clinical centers: HVTN100  Phase I/II randomized, double blind,  placebo controlled, parallel clinical trial, enrolling age range 18-40 of both genders, with primary purpose prevention ... 
The latest WHO and UNAIDS guidelines [WHO  Guideline on when to start antiretroviral therapy and on pre-exposure prophylaxis for HIV. World Health Organization, Geneva; 2015] [UNAIDS  Strategy for 2016—2021: fast-tracking to zero. UNAIDS, Geneva; 2015] are exemplified in a Danish study comprising 20 years follow-up of serodiscordant pairs (man + man in pair, one of them infected) in HIV infected communities. While the pairs were under continuous laboratory check up, the infected persons of them were given antiretroviral drug treatment consecutively, with no skips. Results of the long lasting study were summarized and interpreted by mathematical and epidemiological approaches as well [Okano J.T. et al. (2016):Testing the hypothesis that treatment can eliminate HIV: a nationwide, population-based study of the Danish HIV epidemic in men who have sex with men.  Lancet  Infect. Dis. 16: 789-796.]
It was found that the threshold value to eliminate HIV according to WHO and UNAIDS guidelines {one new HIV infection/1000 person/year}, can be approached by the combined effect of the almost full coverage (92%) in treatment  of infected communities and, the consecutive adherence to antiretroviral therapy ( rate of virus suppression = 98%). The goals were reached by 'treatment as prevention' of HIV infected persons to decrease the risk of HIV transmission to their non-infected partners. Population coverage was further supported by the changes along the 20 years in Danish public health practice. In the period of 1996-2008 the eligible patients for antiretroviral therapy were those with CD4+ lymphocyte number below the threshold of 300/µl. This threshold was reset to level 350/µl in 2008. From 2011 on, irrespective of the CD4+ cell number, all HIV infected persons became eligible for antiretroviral drug therapy. 
Results above highlight the alignment of three optimal contributors to HIV elimination:
  • at individuum level the compliance with, and adherence to treatment,
  • at community level the great coverage (all infected involved in treatment, if possible),
  • supporting biological option is the use of drugs with retarded or no developing resistance to them.

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