Rein Sikut | GlaxoSmithKline, LLP (original) (raw)
Papers by Rein Sikut
Eesti Arst, May 25, 2016
Veel aasta tagasi oli Lääne-Aafrikas puhkenud Ebola-epideemia laienemas, nakatunute arv kahekordi... more Veel aasta tagasi oli Lääne-Aafrikas puhkenud Ebola-epideemia laienemas, nakatunute arv kahekordistus iga kuu ja keegi ei teadnud täpselt, kuhu need näitajad välja võivad jõuda. Epideemia algas juba 2013. aasta lõpul, kuid riigid, kus see algas, viivitasid raporteerimisega kuni 2014. aasta märtsini. Alles 2014. aasta augustis kuulutas Maailma Terviseorganisatsioon välja hädaolukorra ja septembris jõudis kohale suurem rahvusvaheline abi. Veel eelmisel aastal ei olnud ühtegi tõhusat vaktsiini Ebola viiruse vastu ega viirusevastast ravi Ebola viirushaiguse tarvis. Olukord on aga nüüd diametraalselt muutunud. Epideemia on raugenud, Sierra Leone ja Libeeria on juba kuulutatud Ebola viirushaiguse vabaks, Guineas tervenes viimane Ebola-haige novembris 2015 ja aasta lõpul kuulutati ka Guinea Ebola viirushaiguse vabaks. Võrreldes aastataguse olukorraga on Ebola-vastases võitluses nüüd kasutada üks oluline uus vahend: efektiivne vaktsiin. Mitte ühtegi vaktsiini pole varem suudetud nii kiiresti kättesaadavaks teha. Kõikide uute Ebola-juhtumite edaspidisel avastamisel neis riikides rakendatakse kontaktsete isikute kohest vaktsineerimist: märtsis 2016 vaktsineeriti Guineas sel põhjusel üle 1000 inimese.
Vaccine, Apr 1, 2007
A novel animal model for testing the immunogenicity and protective immune response induced by HIV... more A novel animal model for testing the immunogenicity and protective immune response induced by HIV-1 DNA vaccines was developed. DBA/2 mice were immunized with GTU-MultiHIV DNA encoding multigene for Rev, Nef, Tat, optp17/24 and a stretch of Pol/Env epitopes. A single GTU-MultiHIV B-clade specific plasmid or Auxo-GTU-MultiHIV(mix) (mixture of four plasmids with A, B, C and FGH clade specific MultiHIV antigens) were administered via gene gun and cell-mediated and humoral immune responses were analysed. The protective efficacy of the immune response was evaluated by challenging the mice with syngeneic tumor cells (P815) stably transfected with the MultiHIV fusion gene. Our results show that the strong MultiHIV-specific immune response generated by the GTU-MultiHIV vaccines in DBA/2 mice was able to delay the tumor growth substantially, indicating that the CTL response detected in vitro confers protection in vivo. The model described here is a safe and feasible in vivo assay for assessment of the vaccine potency to induce protective cell-mediated immune responses.
Eesti Arst, Nov 28, 2014
Klassikalised põhimõtted vaktsinoloogias formuleeris 19. sajandi lõpul L. Pasteur. See on andnud ... more Klassikalised põhimõtted vaktsinoloogias formuleeris 19. sajandi lõpul L. Pasteur. See on andnud meile vaktsiine 27 haigustekitaja vastu ning viinud paljude nakkushaiguste leviku olulise vähenemiseni, rõugete puhul isegi täieliku kadumiseni. Siiski on hulk üle maailma levivaid haigusi, mille vastu pole suudetud seni kasutatud meetoditega vaktsiini saada. Võib öelda, et klassikalise vaktsinoloogia võimalused on praeguseks ennast ammendanud ja vajadus uute strateegiate järele on ilmselge. Molekulaarbioloogia areng on viimastel aastatel andnud konkreetseid väljundeid uute vaktsiinide saamisel. Genoomika võimalusi kasutades on haigustekitajatest võimalik kiiresti leida seni tundmata antigeene, mis on sobivad vaktsiini tegemiseks. Haigustekitaja genoomi uurimisest saadud info kasutamist vaktsiiniantigeenide leidmiseks nimetatakse kokkuvõtlikult pöördvaktsinoloogiaks. Artiklis on lähemalt vaadeldud, kuidas pöördvaktsinoloogiat kasutati meningokokk B vastase vaktsiini loomisel. Samuti on selgitatud struktuuribioloogia rolli seni kättesaamatuks jäänud vaktsiinide arendamisel või olemasolevate vaktsiinide paremaks muutmisel.
Eesti Arst, May 31, 2012
Autor töötab ettevõtte GlaxoSmithKline teadusnõunikuna ning GSK on üks firmadest, mis toodab ja t... more Autor töötab ettevõtte GlaxoSmithKline teadusnõunikuna ning GSK on üks firmadest, mis toodab ja turustab HIV-ravimeid.
Eesti Arst, May 31, 2013
Möödunud on 30 aastat, mil isoleeriti AIDSi tekitav viirus HIV-1. Molekulaarfülogeneetilised uuri... more Möödunud on 30 aastat, mil isoleeriti AIDSi tekitav viirus HIV-1. Molekulaarfülogeneetilised uuringud näitavad, et HIV-pandeemia sai alguse Aafrika šimpansidel esinevast lähedasest viirusest SIV cpz , mis on inimestele üle kandunud ahvide küttimise ja liha käitlemise käigus ajavahemikul 1902-1921. Üleilmse HIV-pandeemia lähtekohaks peetakse Kongo linna Kinshasat, mis oli 20. sajandi esimesel poolel kiiresti kasvav linn, mis lõi soodsad tingimused viiruse levikuks. Sealt pärinevad ka 2 kõige vanemat HIV-1 sisaldavat koeproovi (1959 ja 1960). HIV leviku esimene sihtkoht väljaspool Aafrikat oli Haiti, sealt edasi jõudis viirus ajavahemikul 1966-1972 USAsse. HIV-patogeneesi mehhanismid on komplekssed: peamine on CD4+ rakkude hävimine, mistõttu tekivad häired nii organismi humoraalse kui ka rakulise immuunsüsteemi toimimisel. Krooniline immuunaktivatsioon on üks tähtsaim tegur immuunpuudulikkuse tekkel. Viiruse isoleerimine ja elutsükli tundmaõppimine võimaldas antiviraalsete ravimite väljatöötamist. Esimene ravim loodi 1987. aastal (NRTI klass), kuid üksinda kasutades oli see väheefektiivne. 1995. aastal loodi uus klass ravimeid (PI) ja alustati kombinatsioonravi mitme toimeainega korraga ning see viis Euroopas AIDSi tekke ja suremuse 10-kordse vähenemiseni. Praegu kasutusel olevad ravimid liigitatakse toimemehhanismi alusel 5 erinevasse klassi ning surmatõvest on saanud ravimite abil kontrolli all hoitav krooniline haigus. Vaatamata pingutustele pole seni suudetud luua vaktsiini, kuid uuringud selles vallas jätkuvad.
Biochemical and Biophysical Research Communications, Sep 1, 1997
CD43 (leukosialin) has hitherto been considered as an exclusive leukocyte marker, but now we repo... more CD43 (leukosialin) has hitherto been considered as an exclusive leukocyte marker, but now we report the expression of CD43 in the epithelial cells of all studied colorectal adenomas (21/21) and in about 50% (18/34) of adenocarcinomas as analyzed both at the mRNA and protein levels. Direct evidence showing the causal role of CD43 in colon tumorigenesis is lacking, but its involvement in leukocyte activation and impairment of apoptotic response suggests a role for CD43 in colon cancer development.
International Journal of Cancer, May 29, 1996
The cancer-associated epitope defined by the monoclonal antibody (MAb) 9H8 was shown to be closel... more The cancer-associated epitope defined by the monoclonal antibody (MAb) 9H8 was shown to be closely related to the T antigen (Thomsen-Friedenreich antigen) by its sensitivity to 0-glycanase treatment of a mucin glycopeptide known to express this epitope. The reactivity with this glycopeptide increased upon neuraminiclase treatment, and among several MAbs tested for ability to block binding of the 9H8 antibody, the one specific for the T antigen was the most efficient. Out of 41 serum samples from breast-cancer patients, 11 showed elevated levels of the 9H8 epitope, and several sera also showed elevated levels of the cancer-associated carbohydrate epitopes sialyl-Lewis a and sialyl-Lewis x. By the use of antibodies specific for the MUC1 apoprotein (Ma552 and HMFG-2) it could be shown that these epitopes were attached to the MUC1 apoprotein in at least 4 of the cases. By combining antibodies specific to 9H8, sialyl-Lewis a and sialyl-Lewis x in catcher and tracer positions in several types of immunofluorometric assays, it was shown that the 9H8 epitope was rarely co-expressed with sialyl-Lewis a or sialyl-Lewis x epitopes an the same molecule, though all were expressed on MUC1 mucins. In fact, they can be considered as mutually exclusive epitopes, suggesting that these sera contained different populations of MUC1 mucins distinguishable by different sets of oligosaccharides. The existence of mutually exclusive carbohydrate epitopes on different MUC1 mucins in one and the same patient should be taken into account when designing immunoassays exploiting MUC1-reactive antibodies.
Tumor Biology, 1998
The 55 antibodies submitted to the ISOBM TD-4 Workshop were analysed for their reactivity with co... more The 55 antibodies submitted to the ISOBM TD-4 Workshop were analysed for their reactivity with core proteins of the heavily glycosylated MUC1 mucins from the colon carcinoma cell line COLO205 and bile. Both these mucins (designated as H-CanAg and SBG2) are highly glycosylated having 15 and 50 sugar residues per oligosaccharide, respectively. Only a few of the antibodies (129, 139, 153 and 162) reacted with both SBG2 and H-CanAg, and not with a control mucin (L-CanAg) having a similar glycosylation as H-CanAg. These antibodies were tested for their ability to catch soluble mucins, and the antibody 162 was found to be good also in this type of assay. The antibodies selected here should be useful for the detection of high glycosylated forms of the MUC1 mucin in tissues and serum.
Cellular Immunology, Mar 1, 1997
teristic high carbohydrate content (usually ú50% of the total mass). Many carcinomas have an abno... more teristic high carbohydrate content (usually ú50% of the total mass). Many carcinomas have an abnormal The effect of two secreted mucin-type glycoproteins increased expression of mucins often with altered caron natural killer (NK) cell cytotoxicity against K562 bohydrate epitopes (2, 3). Mucin-type domains have target cells has been studied. These mucins carry the carcinoma-associated sialyl-Lewis a carbohydrate epi-been shown to be present in most of the characterized topes and were purified from the human colon adenoligands for the selectins having C-type lectin domains, carcinoma cell line COLO 205 secretions, where they indicating an important role of the clustered oligosaclack their cytoplasmic parts. The larger one has an charide chains for binding (4). apoprotein encoded by the MUC1 gene, and the smaller Recent studies have shown that several membrane one has CD43 (leukosialin) as the core protein. The proteins expressed on natural killer (NK) cells have purified MUC1 mucin could inhibit the target cell lysis sequences homologous to the C-type lectins. This is the by NK cells in a dose-response-dependent way, case for both the inhibitory murine MHC-recognizing whereas other mucin domains of similar size showed receptors such as the Ly-49 family, as well as for targetno inhibition. The second mucin, CD43, inhibited lysis recognizing human molecules such as NKR-P1 (5). The by NK cells, although less than the larger one. The former type of receptors is binding to the MHC mole-MUC1 mucin bound to the enriched natural killer cell cule where one of its N-linked oligosaccharides is probpreparations in a partial Ca 2/-dependent way as well. ably involved. Much less is known and understood This mucin also bound to the target cells. The K562 about the second group of C-type lectin receptors probacells, normally expressing high amount of CD43, showed an increased resistance to lysis by NK cells bly being part of NK cell-specific triggering events. One when transfected with MUC1 cDNA compared with possible role could be to detect carcinoma-associated nontransfected cells. One can speculate that mucins and diverse carbohydrate ligands produced by tumor secreted or expressed in the plasma membrane of cancells (6). An understanding of the role of the receptors cer cells could interfere with NK cell-mediated lysis. with a C-type lectin domain on NK cells and identifica-᭧ 1997 Academic Press tion of their possible oligosaccharide ligands should help to elucidate the mechanisms involved in the cascade of events resulting in the killing of target cells and INTRODUCTION in the immune surveillance of cancer. The occurrence of clinical tumors involves, as one out of many reasons, Mucins are a group of large, highly O-glycosylated the failure of NK cells to eliminate the tumor cells beglycoproteins that appear in secreted or membranecause of weakened NK cell response. An explanation bound form covering the respiratory, gastrointestinal, for this could be failing effector cells or a selection of and urogenital tracts (1, 2). The apoproteins of typical tumor cells using various mechanisms to escape the mucins are characterized by domains rich in the amino NK cell system. If oligosaccharides of the target cell acids serine, threonine, and proline where the hydroxy are of importance for the NK cell recognition, these are amino acids are attachment sites for O-glycans (mucinexpected to have different effects depending on their type domain). These domains give mucins their characstructure. Another important issue is the potential effect of secreted ligands. Glycoproteins with mucin-type
AIDS Research and Human Retroviruses, Jul 1, 2006
A multiHIV fusion gene expressing an antigenic fusion protein composed of regulatory HIV-1 protei... more A multiHIV fusion gene expressing an antigenic fusion protein composed of regulatory HIV-1 proteins Rev, Nef, and Tat, as well as Gag p17/p24 and a stretch of 11 cytotoxic T lymphocyte (CTL) epitope clusters from Pol and Env, was cloned into a novel DNA vector named the Gene Transport Unit (GTU). A mouse H-2(d)-restricted HIV-1 gp120 epitope (RGPGRAFVTI) was cloned into the fusion gene as well. In addition to the HIV- 1 genes the GTU codes for a nuclear anchoring protein (bovine papilloma virus E2), ensuring the long maintenance of the vector and a high expression level of the selected immunogens. BALB/c mice were immunized with the GTU-MultiHIV DNA construct by different routes and regimens of immunization to assess the immunogenicity of the DNA vaccine in vivo. Mice developed strong CD8(+) CTL responses to HIV-1 Env and Gag measured by an ELISPOT-IFN-gamma assay and chromium release assay. In addition, T cell responses to regulatory proteins Rev, Nef, and Tat were induced. Antibody responses were detected to each of the HIV antigens encoded by the DNA construct. Minimal doses of the GTU-MultiHIV DNA delivered by gene gun were potent in inducing significant HIV-specific CTL responses. The equivalent doses of the conventional plasmid expressing MultiHIV DNA delivered by gene gun failed to do so. An ideal DNA vaccine should yield high expression of the viral antigens for a prolonged period of time, and expression of the multiple viral antigens is probably required for the induction of a broad and protective immune response. The GTU-MultiHIV DNA vaccine described is a good vaccine candidate that meets the above criteria.
Tumor Biology, 1992
A new monoclonal antibody (MAb 9H8, IgM class) reactive with human ovarian carcinoma has been rai... more A new monoclonal antibody (MAb 9H8, IgM class) reactive with human ovarian carcinoma has been raised after immunizing C57BL/6 mice with bovine sperm. Immunohistological studies indicated that 20/21 serous ovarian adenocarcinomas expressed 9H8-defined antigen but it was absent in benign ovarian tumors (0/11). 1/11 of breast carcinomas and 5/5 of rectal carcinomas expressed this antigen, although to a considerably lesser degree. Tumors of lung, skin, brain and mesothelium were negative. The antigen was also expressed in embryonic skin, in renal collecting tubule cells and in saliva. In bovine, human and mouse sperm the antigen is confined to the acrosomal region. The molecular weight of this antigen was determined by Western blot analysis and gel filtration. In SDS-PAGE the antigen ran as a broad band barely entering the 7% gel, indicating an apparent molecular weight > 300 kDa. In the absence of detergents and reducing agents this glycoprotein forms larger complexes (> 1,500 kDa) as determined by gel filtration on Sephacryl S300. The epitope contains carbohydrate structures recognized by lectin PNA (peanut agglutinin).
PLOS Pathogens, Sep 5, 2013
<p>(A) COP-5 cells were transfected with siRNAs against MDA-5, RIG-I, and LGP2 or combinati... more <p>(A) COP-5 cells were transfected with siRNAs against MDA-5, RIG-I, and LGP2 or combinations thereof. After 48 hr, cells were transfected with poly(I:C) dsRNA, and after 24 hr, the amount of secreted IFN-β was measured by ELISA (upper panel). The efficiency of RLR protein knockdown was assessed by immunoblot assay (lower panel). Cells lysates were separated by SDS-PAGE and immunoblotted with different antibodies. Neg. ctrl., negative control non-targeting siRNA; mock, transfection without siRNA. (B) COP-5 cells were transfected with siRNAs as described in (A). After 48 hr, cells were transfected with pRep-RDR plasmid DNA, and after 48 hr, the amount of secreted IFN-β was measured as described in (A) (upper panel). The RLR knockdown efficiency (lower panel) was assessed as described in (A). Expression of SFV replicase was analyzed using antibodies against nsP4 and nsP2; ACTIN was used as loading control. (C and D) MEFs were transfected with siRNAs as described in (A). After 48 hr, cells were infected with SFV4-Rluc-RDR at different MOIs. After an additional 12 hr, the amount of IFN-β was measured (C) and cell lysates were prepared for the Renilla luciferase reporter assay (D). Immunoblots (A and B) and panels (C and D) are representative examples of two independent experiments. Error bars (A and B) represent the standard deviation of three experiments. ***p<0.001 (t-test).</p
PLOS Pathogens, Sep 5, 2013
Type I interferons (IFN) are important for antiviral responses. Melanoma differentiation-associat... more Type I interferons (IFN) are important for antiviral responses. Melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-induced gene I (RIG-I) proteins detect cytosolic double-stranded RNA (dsRNA) or 59-triphosphate (59-ppp) RNA and mediate IFN production. Cytosolic 59-ppp RNA and dsRNA are generated during viral RNA replication and transcription by viral RNA replicases [RNA-dependent RNA polymerases (RdRp)]. Here, we show that the Semliki Forest virus (SFV) RNA replicase can induce IFN-b independently of viral RNA replication and transcription. The SFV replicase converts host cell RNA into 59-ppp dsRNA and induces IFN-b through the RIG-I and MDA-5 pathways. Inactivation of the SFV replicase RdRp activity prevents IFN-b induction. These IFN-inducing modified host cell RNAs are abundantly produced during both wild-type SFV and its non-pathogenic mutant infection. Furthermore, in contrast to the wild-type SFV replicase a non-pathogenic mutant replicase triggers increased IFN-b production, which leads to a shutdown of virus replication. These results suggest that host cells can restrict RNA virus replication by detecting the products of unspecific viral replicase RdRp activity.
Ophthalmic Research, 1994
All 47 human senile cataractous lenses studied by a direct immunohistochemical method for the pre... more All 47 human senile cataractous lenses studied by a direct immunohistochemical method for the presence of immunoglobulins yielded negative results. Consequently, cataract cannot be an autoimmune disease as has been suggested by some authors.
Antiretroviirusravimite väljatöötamine on olnud märkimisväärselt kiire ja praeguseks on jõudnud k... more Antiretroviirusravimite väljatöötamine on olnud märkimisväärselt kiire ja praeguseks on jõudnud kasutusse juba 31 toimeainet, mis oma toimemehhanismide alusel jagunevad 5 ravimiklassi. Uued ravimid on tavaliselt üks kord päevas manustatavad, neil on vähem kõrvaltoimeid ja väiksem võimalus ravimiresistentsuse tekkeks. Uute toimeainete abil on ravi efektiivsus (viirussupressiooni saavutanud patsientide osakaal 48. nädalaks pärast ravi alustamist) tõusnud üle 90%. Tänu uutele toimeainetele on lihtsustumas raviskeemid ja käesoleval aastal on esimest korda lubatud kasutusse kahest toimeainest koosnev tablett, mis on mõeldud viiruskontrolli pikaajaliseks säilitamiseks. Arendusjärgus on veel mitmed uued ravimid, sealhulgas pikatoimelised süstitavad ravimid
Möödunud on 30 aastat, mil isoleeriti AIDSi tekitav viirus HIV-1. Molekulaarfülogenee-tilised uur... more Möödunud on 30 aastat, mil isoleeriti AIDSi tekitav viirus HIV-1. Molekulaarfülogenee-tilised uuringud näitavad, et HIV-pandeemia sai alguse Aafrika šimpansidel esinevast lähedasest viirusest SIV cpz , mis on inimestele üle kandunud ahvide küttimise ja liha käitlemise käigus ajavahemikul 1902-1921. Üleilmse HIV-pandeemia lähtekohaks peetakse Kongo linna Kinshasat, mis oli 20. sajandi esimesel poolel kiiresti kasvav linn, mis lõi soodsad tingimused viiruse levikuks. Sealt pärinevad ka 2 kõige vanemat HIV-1 sisaldavat koeproovi (1959 ja 1960). HIV leviku esimene sihtkoht väljaspool Aafrikat oli Haiti, sealt edasi jõudis viirus ajavahemikul 1966-1972 USAsse. HIV-patogeneesi mehhanismid on komplekssed: peamine on CD4+ rakkude hävimine, mistõttu tekivad häired nii organismi humoraalse kui ka rakulise immuunsüsteemi toimimisel. Kroo-niline immuunaktivatsioon on üks tähtsaim tegur immuunpuudulikkuse tekkel. Viiruse isoleerimine ja elutsükli tundmaõppimine võimaldas antiviraalsete ravimite ...
Moodunud on 30 aastat, mil isoleeriti AIDSi tekitav viirus HIV-1. Molekulaarfulogeneetilised uuri... more Moodunud on 30 aastat, mil isoleeriti AIDSi tekitav viirus HIV-1. Molekulaarfulogeneetilised uuringud naitavad, et HIV-pandeemia sai alguse Aafrika simpansidel esinevastlahedasest viirusest SIVcpz, mis on inimestele ule kandunud ahvide kuttimise ja lihakaitlemise kaigus ajavahemikul 1902–1921. Uleilmse HIV-pandeemia lahtekohaks peetakse Kongo linna Kinshasat, mis oli 20. sajandi esimesel poolel kiiresti kasvav linn, mis loi soodsad tingimused viiruse levikuks. Sealt parinevad ka 2 koige vanemat HIV-1 sisaldavat koeproovi (1959 ja 1960). HIV leviku esimene sihtkoht valjaspool Aafrikat oli Haiti, sealt edasi joudis viirus ajavahemikul 1966–1972 USAsse. HIV-patogeneesi mehhanismid on komplekssed: peamine on CD4+ rakkude havimine, mistottu tekivad haired nii organismi humoraalse kui ka rakulise immuunsusteemi toimimisel. Krooniline immuunaktivatsioon on uks tahtsaim tegur immuunpuudulikkuse tekkel. Viiruse isoleerimine ja elutsukli tundmaoppimine voimaldas antiviraalsete ravimite valjat...
Eesti Arst, May 25, 2016
Veel aasta tagasi oli Lääne-Aafrikas puhkenud Ebola-epideemia laienemas, nakatunute arv kahekordi... more Veel aasta tagasi oli Lääne-Aafrikas puhkenud Ebola-epideemia laienemas, nakatunute arv kahekordistus iga kuu ja keegi ei teadnud täpselt, kuhu need näitajad välja võivad jõuda. Epideemia algas juba 2013. aasta lõpul, kuid riigid, kus see algas, viivitasid raporteerimisega kuni 2014. aasta märtsini. Alles 2014. aasta augustis kuulutas Maailma Terviseorganisatsioon välja hädaolukorra ja septembris jõudis kohale suurem rahvusvaheline abi. Veel eelmisel aastal ei olnud ühtegi tõhusat vaktsiini Ebola viiruse vastu ega viirusevastast ravi Ebola viirushaiguse tarvis. Olukord on aga nüüd diametraalselt muutunud. Epideemia on raugenud, Sierra Leone ja Libeeria on juba kuulutatud Ebola viirushaiguse vabaks, Guineas tervenes viimane Ebola-haige novembris 2015 ja aasta lõpul kuulutati ka Guinea Ebola viirushaiguse vabaks. Võrreldes aastataguse olukorraga on Ebola-vastases võitluses nüüd kasutada üks oluline uus vahend: efektiivne vaktsiin. Mitte ühtegi vaktsiini pole varem suudetud nii kiiresti kättesaadavaks teha. Kõikide uute Ebola-juhtumite edaspidisel avastamisel neis riikides rakendatakse kontaktsete isikute kohest vaktsineerimist: märtsis 2016 vaktsineeriti Guineas sel põhjusel üle 1000 inimese.
Vaccine, Apr 1, 2007
A novel animal model for testing the immunogenicity and protective immune response induced by HIV... more A novel animal model for testing the immunogenicity and protective immune response induced by HIV-1 DNA vaccines was developed. DBA/2 mice were immunized with GTU-MultiHIV DNA encoding multigene for Rev, Nef, Tat, optp17/24 and a stretch of Pol/Env epitopes. A single GTU-MultiHIV B-clade specific plasmid or Auxo-GTU-MultiHIV(mix) (mixture of four plasmids with A, B, C and FGH clade specific MultiHIV antigens) were administered via gene gun and cell-mediated and humoral immune responses were analysed. The protective efficacy of the immune response was evaluated by challenging the mice with syngeneic tumor cells (P815) stably transfected with the MultiHIV fusion gene. Our results show that the strong MultiHIV-specific immune response generated by the GTU-MultiHIV vaccines in DBA/2 mice was able to delay the tumor growth substantially, indicating that the CTL response detected in vitro confers protection in vivo. The model described here is a safe and feasible in vivo assay for assessment of the vaccine potency to induce protective cell-mediated immune responses.
Eesti Arst, Nov 28, 2014
Klassikalised põhimõtted vaktsinoloogias formuleeris 19. sajandi lõpul L. Pasteur. See on andnud ... more Klassikalised põhimõtted vaktsinoloogias formuleeris 19. sajandi lõpul L. Pasteur. See on andnud meile vaktsiine 27 haigustekitaja vastu ning viinud paljude nakkushaiguste leviku olulise vähenemiseni, rõugete puhul isegi täieliku kadumiseni. Siiski on hulk üle maailma levivaid haigusi, mille vastu pole suudetud seni kasutatud meetoditega vaktsiini saada. Võib öelda, et klassikalise vaktsinoloogia võimalused on praeguseks ennast ammendanud ja vajadus uute strateegiate järele on ilmselge. Molekulaarbioloogia areng on viimastel aastatel andnud konkreetseid väljundeid uute vaktsiinide saamisel. Genoomika võimalusi kasutades on haigustekitajatest võimalik kiiresti leida seni tundmata antigeene, mis on sobivad vaktsiini tegemiseks. Haigustekitaja genoomi uurimisest saadud info kasutamist vaktsiiniantigeenide leidmiseks nimetatakse kokkuvõtlikult pöördvaktsinoloogiaks. Artiklis on lähemalt vaadeldud, kuidas pöördvaktsinoloogiat kasutati meningokokk B vastase vaktsiini loomisel. Samuti on selgitatud struktuuribioloogia rolli seni kättesaamatuks jäänud vaktsiinide arendamisel või olemasolevate vaktsiinide paremaks muutmisel.
Eesti Arst, May 31, 2012
Autor töötab ettevõtte GlaxoSmithKline teadusnõunikuna ning GSK on üks firmadest, mis toodab ja t... more Autor töötab ettevõtte GlaxoSmithKline teadusnõunikuna ning GSK on üks firmadest, mis toodab ja turustab HIV-ravimeid.
Eesti Arst, May 31, 2013
Möödunud on 30 aastat, mil isoleeriti AIDSi tekitav viirus HIV-1. Molekulaarfülogeneetilised uuri... more Möödunud on 30 aastat, mil isoleeriti AIDSi tekitav viirus HIV-1. Molekulaarfülogeneetilised uuringud näitavad, et HIV-pandeemia sai alguse Aafrika šimpansidel esinevast lähedasest viirusest SIV cpz , mis on inimestele üle kandunud ahvide küttimise ja liha käitlemise käigus ajavahemikul 1902-1921. Üleilmse HIV-pandeemia lähtekohaks peetakse Kongo linna Kinshasat, mis oli 20. sajandi esimesel poolel kiiresti kasvav linn, mis lõi soodsad tingimused viiruse levikuks. Sealt pärinevad ka 2 kõige vanemat HIV-1 sisaldavat koeproovi (1959 ja 1960). HIV leviku esimene sihtkoht väljaspool Aafrikat oli Haiti, sealt edasi jõudis viirus ajavahemikul 1966-1972 USAsse. HIV-patogeneesi mehhanismid on komplekssed: peamine on CD4+ rakkude hävimine, mistõttu tekivad häired nii organismi humoraalse kui ka rakulise immuunsüsteemi toimimisel. Krooniline immuunaktivatsioon on üks tähtsaim tegur immuunpuudulikkuse tekkel. Viiruse isoleerimine ja elutsükli tundmaõppimine võimaldas antiviraalsete ravimite väljatöötamist. Esimene ravim loodi 1987. aastal (NRTI klass), kuid üksinda kasutades oli see väheefektiivne. 1995. aastal loodi uus klass ravimeid (PI) ja alustati kombinatsioonravi mitme toimeainega korraga ning see viis Euroopas AIDSi tekke ja suremuse 10-kordse vähenemiseni. Praegu kasutusel olevad ravimid liigitatakse toimemehhanismi alusel 5 erinevasse klassi ning surmatõvest on saanud ravimite abil kontrolli all hoitav krooniline haigus. Vaatamata pingutustele pole seni suudetud luua vaktsiini, kuid uuringud selles vallas jätkuvad.
Biochemical and Biophysical Research Communications, Sep 1, 1997
CD43 (leukosialin) has hitherto been considered as an exclusive leukocyte marker, but now we repo... more CD43 (leukosialin) has hitherto been considered as an exclusive leukocyte marker, but now we report the expression of CD43 in the epithelial cells of all studied colorectal adenomas (21/21) and in about 50% (18/34) of adenocarcinomas as analyzed both at the mRNA and protein levels. Direct evidence showing the causal role of CD43 in colon tumorigenesis is lacking, but its involvement in leukocyte activation and impairment of apoptotic response suggests a role for CD43 in colon cancer development.
International Journal of Cancer, May 29, 1996
The cancer-associated epitope defined by the monoclonal antibody (MAb) 9H8 was shown to be closel... more The cancer-associated epitope defined by the monoclonal antibody (MAb) 9H8 was shown to be closely related to the T antigen (Thomsen-Friedenreich antigen) by its sensitivity to 0-glycanase treatment of a mucin glycopeptide known to express this epitope. The reactivity with this glycopeptide increased upon neuraminiclase treatment, and among several MAbs tested for ability to block binding of the 9H8 antibody, the one specific for the T antigen was the most efficient. Out of 41 serum samples from breast-cancer patients, 11 showed elevated levels of the 9H8 epitope, and several sera also showed elevated levels of the cancer-associated carbohydrate epitopes sialyl-Lewis a and sialyl-Lewis x. By the use of antibodies specific for the MUC1 apoprotein (Ma552 and HMFG-2) it could be shown that these epitopes were attached to the MUC1 apoprotein in at least 4 of the cases. By combining antibodies specific to 9H8, sialyl-Lewis a and sialyl-Lewis x in catcher and tracer positions in several types of immunofluorometric assays, it was shown that the 9H8 epitope was rarely co-expressed with sialyl-Lewis a or sialyl-Lewis x epitopes an the same molecule, though all were expressed on MUC1 mucins. In fact, they can be considered as mutually exclusive epitopes, suggesting that these sera contained different populations of MUC1 mucins distinguishable by different sets of oligosaccharides. The existence of mutually exclusive carbohydrate epitopes on different MUC1 mucins in one and the same patient should be taken into account when designing immunoassays exploiting MUC1-reactive antibodies.
Tumor Biology, 1998
The 55 antibodies submitted to the ISOBM TD-4 Workshop were analysed for their reactivity with co... more The 55 antibodies submitted to the ISOBM TD-4 Workshop were analysed for their reactivity with core proteins of the heavily glycosylated MUC1 mucins from the colon carcinoma cell line COLO205 and bile. Both these mucins (designated as H-CanAg and SBG2) are highly glycosylated having 15 and 50 sugar residues per oligosaccharide, respectively. Only a few of the antibodies (129, 139, 153 and 162) reacted with both SBG2 and H-CanAg, and not with a control mucin (L-CanAg) having a similar glycosylation as H-CanAg. These antibodies were tested for their ability to catch soluble mucins, and the antibody 162 was found to be good also in this type of assay. The antibodies selected here should be useful for the detection of high glycosylated forms of the MUC1 mucin in tissues and serum.
Cellular Immunology, Mar 1, 1997
teristic high carbohydrate content (usually ú50% of the total mass). Many carcinomas have an abno... more teristic high carbohydrate content (usually ú50% of the total mass). Many carcinomas have an abnormal The effect of two secreted mucin-type glycoproteins increased expression of mucins often with altered caron natural killer (NK) cell cytotoxicity against K562 bohydrate epitopes (2, 3). Mucin-type domains have target cells has been studied. These mucins carry the carcinoma-associated sialyl-Lewis a carbohydrate epi-been shown to be present in most of the characterized topes and were purified from the human colon adenoligands for the selectins having C-type lectin domains, carcinoma cell line COLO 205 secretions, where they indicating an important role of the clustered oligosaclack their cytoplasmic parts. The larger one has an charide chains for binding (4). apoprotein encoded by the MUC1 gene, and the smaller Recent studies have shown that several membrane one has CD43 (leukosialin) as the core protein. The proteins expressed on natural killer (NK) cells have purified MUC1 mucin could inhibit the target cell lysis sequences homologous to the C-type lectins. This is the by NK cells in a dose-response-dependent way, case for both the inhibitory murine MHC-recognizing whereas other mucin domains of similar size showed receptors such as the Ly-49 family, as well as for targetno inhibition. The second mucin, CD43, inhibited lysis recognizing human molecules such as NKR-P1 (5). The by NK cells, although less than the larger one. The former type of receptors is binding to the MHC mole-MUC1 mucin bound to the enriched natural killer cell cule where one of its N-linked oligosaccharides is probpreparations in a partial Ca 2/-dependent way as well. ably involved. Much less is known and understood This mucin also bound to the target cells. The K562 about the second group of C-type lectin receptors probacells, normally expressing high amount of CD43, showed an increased resistance to lysis by NK cells bly being part of NK cell-specific triggering events. One when transfected with MUC1 cDNA compared with possible role could be to detect carcinoma-associated nontransfected cells. One can speculate that mucins and diverse carbohydrate ligands produced by tumor secreted or expressed in the plasma membrane of cancells (6). An understanding of the role of the receptors cer cells could interfere with NK cell-mediated lysis. with a C-type lectin domain on NK cells and identifica-᭧ 1997 Academic Press tion of their possible oligosaccharide ligands should help to elucidate the mechanisms involved in the cascade of events resulting in the killing of target cells and INTRODUCTION in the immune surveillance of cancer. The occurrence of clinical tumors involves, as one out of many reasons, Mucins are a group of large, highly O-glycosylated the failure of NK cells to eliminate the tumor cells beglycoproteins that appear in secreted or membranecause of weakened NK cell response. An explanation bound form covering the respiratory, gastrointestinal, for this could be failing effector cells or a selection of and urogenital tracts (1, 2). The apoproteins of typical tumor cells using various mechanisms to escape the mucins are characterized by domains rich in the amino NK cell system. If oligosaccharides of the target cell acids serine, threonine, and proline where the hydroxy are of importance for the NK cell recognition, these are amino acids are attachment sites for O-glycans (mucinexpected to have different effects depending on their type domain). These domains give mucins their characstructure. Another important issue is the potential effect of secreted ligands. Glycoproteins with mucin-type
AIDS Research and Human Retroviruses, Jul 1, 2006
A multiHIV fusion gene expressing an antigenic fusion protein composed of regulatory HIV-1 protei... more A multiHIV fusion gene expressing an antigenic fusion protein composed of regulatory HIV-1 proteins Rev, Nef, and Tat, as well as Gag p17/p24 and a stretch of 11 cytotoxic T lymphocyte (CTL) epitope clusters from Pol and Env, was cloned into a novel DNA vector named the Gene Transport Unit (GTU). A mouse H-2(d)-restricted HIV-1 gp120 epitope (RGPGRAFVTI) was cloned into the fusion gene as well. In addition to the HIV- 1 genes the GTU codes for a nuclear anchoring protein (bovine papilloma virus E2), ensuring the long maintenance of the vector and a high expression level of the selected immunogens. BALB/c mice were immunized with the GTU-MultiHIV DNA construct by different routes and regimens of immunization to assess the immunogenicity of the DNA vaccine in vivo. Mice developed strong CD8(+) CTL responses to HIV-1 Env and Gag measured by an ELISPOT-IFN-gamma assay and chromium release assay. In addition, T cell responses to regulatory proteins Rev, Nef, and Tat were induced. Antibody responses were detected to each of the HIV antigens encoded by the DNA construct. Minimal doses of the GTU-MultiHIV DNA delivered by gene gun were potent in inducing significant HIV-specific CTL responses. The equivalent doses of the conventional plasmid expressing MultiHIV DNA delivered by gene gun failed to do so. An ideal DNA vaccine should yield high expression of the viral antigens for a prolonged period of time, and expression of the multiple viral antigens is probably required for the induction of a broad and protective immune response. The GTU-MultiHIV DNA vaccine described is a good vaccine candidate that meets the above criteria.
Tumor Biology, 1992
A new monoclonal antibody (MAb 9H8, IgM class) reactive with human ovarian carcinoma has been rai... more A new monoclonal antibody (MAb 9H8, IgM class) reactive with human ovarian carcinoma has been raised after immunizing C57BL/6 mice with bovine sperm. Immunohistological studies indicated that 20/21 serous ovarian adenocarcinomas expressed 9H8-defined antigen but it was absent in benign ovarian tumors (0/11). 1/11 of breast carcinomas and 5/5 of rectal carcinomas expressed this antigen, although to a considerably lesser degree. Tumors of lung, skin, brain and mesothelium were negative. The antigen was also expressed in embryonic skin, in renal collecting tubule cells and in saliva. In bovine, human and mouse sperm the antigen is confined to the acrosomal region. The molecular weight of this antigen was determined by Western blot analysis and gel filtration. In SDS-PAGE the antigen ran as a broad band barely entering the 7% gel, indicating an apparent molecular weight > 300 kDa. In the absence of detergents and reducing agents this glycoprotein forms larger complexes (> 1,500 kDa) as determined by gel filtration on Sephacryl S300. The epitope contains carbohydrate structures recognized by lectin PNA (peanut agglutinin).
PLOS Pathogens, Sep 5, 2013
<p>(A) COP-5 cells were transfected with siRNAs against MDA-5, RIG-I, and LGP2 or combinati... more <p>(A) COP-5 cells were transfected with siRNAs against MDA-5, RIG-I, and LGP2 or combinations thereof. After 48 hr, cells were transfected with poly(I:C) dsRNA, and after 24 hr, the amount of secreted IFN-β was measured by ELISA (upper panel). The efficiency of RLR protein knockdown was assessed by immunoblot assay (lower panel). Cells lysates were separated by SDS-PAGE and immunoblotted with different antibodies. Neg. ctrl., negative control non-targeting siRNA; mock, transfection without siRNA. (B) COP-5 cells were transfected with siRNAs as described in (A). After 48 hr, cells were transfected with pRep-RDR plasmid DNA, and after 48 hr, the amount of secreted IFN-β was measured as described in (A) (upper panel). The RLR knockdown efficiency (lower panel) was assessed as described in (A). Expression of SFV replicase was analyzed using antibodies against nsP4 and nsP2; ACTIN was used as loading control. (C and D) MEFs were transfected with siRNAs as described in (A). After 48 hr, cells were infected with SFV4-Rluc-RDR at different MOIs. After an additional 12 hr, the amount of IFN-β was measured (C) and cell lysates were prepared for the Renilla luciferase reporter assay (D). Immunoblots (A and B) and panels (C and D) are representative examples of two independent experiments. Error bars (A and B) represent the standard deviation of three experiments. ***p<0.001 (t-test).</p
PLOS Pathogens, Sep 5, 2013
Type I interferons (IFN) are important for antiviral responses. Melanoma differentiation-associat... more Type I interferons (IFN) are important for antiviral responses. Melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-induced gene I (RIG-I) proteins detect cytosolic double-stranded RNA (dsRNA) or 59-triphosphate (59-ppp) RNA and mediate IFN production. Cytosolic 59-ppp RNA and dsRNA are generated during viral RNA replication and transcription by viral RNA replicases [RNA-dependent RNA polymerases (RdRp)]. Here, we show that the Semliki Forest virus (SFV) RNA replicase can induce IFN-b independently of viral RNA replication and transcription. The SFV replicase converts host cell RNA into 59-ppp dsRNA and induces IFN-b through the RIG-I and MDA-5 pathways. Inactivation of the SFV replicase RdRp activity prevents IFN-b induction. These IFN-inducing modified host cell RNAs are abundantly produced during both wild-type SFV and its non-pathogenic mutant infection. Furthermore, in contrast to the wild-type SFV replicase a non-pathogenic mutant replicase triggers increased IFN-b production, which leads to a shutdown of virus replication. These results suggest that host cells can restrict RNA virus replication by detecting the products of unspecific viral replicase RdRp activity.
Ophthalmic Research, 1994
All 47 human senile cataractous lenses studied by a direct immunohistochemical method for the pre... more All 47 human senile cataractous lenses studied by a direct immunohistochemical method for the presence of immunoglobulins yielded negative results. Consequently, cataract cannot be an autoimmune disease as has been suggested by some authors.
Antiretroviirusravimite väljatöötamine on olnud märkimisväärselt kiire ja praeguseks on jõudnud k... more Antiretroviirusravimite väljatöötamine on olnud märkimisväärselt kiire ja praeguseks on jõudnud kasutusse juba 31 toimeainet, mis oma toimemehhanismide alusel jagunevad 5 ravimiklassi. Uued ravimid on tavaliselt üks kord päevas manustatavad, neil on vähem kõrvaltoimeid ja väiksem võimalus ravimiresistentsuse tekkeks. Uute toimeainete abil on ravi efektiivsus (viirussupressiooni saavutanud patsientide osakaal 48. nädalaks pärast ravi alustamist) tõusnud üle 90%. Tänu uutele toimeainetele on lihtsustumas raviskeemid ja käesoleval aastal on esimest korda lubatud kasutusse kahest toimeainest koosnev tablett, mis on mõeldud viiruskontrolli pikaajaliseks säilitamiseks. Arendusjärgus on veel mitmed uued ravimid, sealhulgas pikatoimelised süstitavad ravimid
Möödunud on 30 aastat, mil isoleeriti AIDSi tekitav viirus HIV-1. Molekulaarfülogenee-tilised uur... more Möödunud on 30 aastat, mil isoleeriti AIDSi tekitav viirus HIV-1. Molekulaarfülogenee-tilised uuringud näitavad, et HIV-pandeemia sai alguse Aafrika šimpansidel esinevast lähedasest viirusest SIV cpz , mis on inimestele üle kandunud ahvide küttimise ja liha käitlemise käigus ajavahemikul 1902-1921. Üleilmse HIV-pandeemia lähtekohaks peetakse Kongo linna Kinshasat, mis oli 20. sajandi esimesel poolel kiiresti kasvav linn, mis lõi soodsad tingimused viiruse levikuks. Sealt pärinevad ka 2 kõige vanemat HIV-1 sisaldavat koeproovi (1959 ja 1960). HIV leviku esimene sihtkoht väljaspool Aafrikat oli Haiti, sealt edasi jõudis viirus ajavahemikul 1966-1972 USAsse. HIV-patogeneesi mehhanismid on komplekssed: peamine on CD4+ rakkude hävimine, mistõttu tekivad häired nii organismi humoraalse kui ka rakulise immuunsüsteemi toimimisel. Kroo-niline immuunaktivatsioon on üks tähtsaim tegur immuunpuudulikkuse tekkel. Viiruse isoleerimine ja elutsükli tundmaõppimine võimaldas antiviraalsete ravimite ...
Moodunud on 30 aastat, mil isoleeriti AIDSi tekitav viirus HIV-1. Molekulaarfulogeneetilised uuri... more Moodunud on 30 aastat, mil isoleeriti AIDSi tekitav viirus HIV-1. Molekulaarfulogeneetilised uuringud naitavad, et HIV-pandeemia sai alguse Aafrika simpansidel esinevastlahedasest viirusest SIVcpz, mis on inimestele ule kandunud ahvide kuttimise ja lihakaitlemise kaigus ajavahemikul 1902–1921. Uleilmse HIV-pandeemia lahtekohaks peetakse Kongo linna Kinshasat, mis oli 20. sajandi esimesel poolel kiiresti kasvav linn, mis loi soodsad tingimused viiruse levikuks. Sealt parinevad ka 2 koige vanemat HIV-1 sisaldavat koeproovi (1959 ja 1960). HIV leviku esimene sihtkoht valjaspool Aafrikat oli Haiti, sealt edasi joudis viirus ajavahemikul 1966–1972 USAsse. HIV-patogeneesi mehhanismid on komplekssed: peamine on CD4+ rakkude havimine, mistottu tekivad haired nii organismi humoraalse kui ka rakulise immuunsusteemi toimimisel. Krooniline immuunaktivatsioon on uks tahtsaim tegur immuunpuudulikkuse tekkel. Viiruse isoleerimine ja elutsukli tundmaoppimine voimaldas antiviraalsete ravimite valjat...