Development and determination of immunogenicity of an mRNA vaccine expressing GRA1 protein against Toxoplasma gondii

Project no. 32019 · Status: Kapanmış · 2024

Summary

<p>Toxoplasma&nbsp;gondii&nbsp;is&nbsp;an&nbsp;obligate&nbsp;intracellular&nbsp;parasite&nbsp;that&nbsp;infects&nbsp;many&nbsp;animals&nbsp;besides&nbsp;humans,&nbsp;causing&nbsp;toxoplasmosis.&nbsp;It&nbsp;is&nbsp;estimated&nbsp;that&nbsp;approximately&nbsp;one-third&nbsp;of&nbsp;the&nbsp;human&nbsp;population&nbsp;in&nbsp;worldwide&nbsp;is&nbsp;infected&nbsp;with&nbsp;T.&nbsp;gondii.&nbsp;T.&nbsp;gondii&nbsp;can&nbsp;cause&nbsp;miscarriage&nbsp;or&nbsp;malformations&nbsp;in&nbsp;pregnant&nbsp;women,&nbsp;while&nbsp;it&nbsp;can&nbsp;cause&nbsp;diseases&nbsp;such&nbsp;as&nbsp;encephalitis,&nbsp;pneumonia&nbsp;or&nbsp;myocarditis&nbsp;in&nbsp;patients&nbsp;with&nbsp;suppressed&nbsp;immune&nbsp;systems.&nbsp;In&nbsp;addition,&nbsp;recent&nbsp;studies&nbsp;have&nbsp;established&nbsp;a&nbsp;relationship&nbsp;between&nbsp;toxoplasmosis&nbsp;and&nbsp;psychiatric&nbsp;diseases.&nbsp;T.&nbsp;gondii&nbsp;causes&nbsp;serious&nbsp;economic&nbsp;losses,&nbsp;especially&nbsp;in&nbsp;sheep,&nbsp;by&nbsp;causing&nbsp;abortion.&nbsp;Drugs&nbsp;used&nbsp;against&nbsp;the&nbsp;disease&nbsp;have&nbsp;serious&nbsp;side&nbsp;effects&nbsp;and&nbsp;are&nbsp;ineffective&nbsp;against&nbsp;chronic&nbsp;toxoplasmosis.&nbsp;One&nbsp;of&nbsp;the&nbsp;most&nbsp;effective&nbsp;ways&nbsp;to&nbsp;prevent&nbsp;such&nbsp;negative&nbsp;effects&nbsp;is&nbsp;vaccination,&nbsp;but&nbsp;there&nbsp;is&nbsp;no&nbsp;vaccine&nbsp;approved&nbsp;for&nbsp;human&nbsp;use&nbsp;yet.&nbsp;Therefore,&nbsp;within&nbsp;the&nbsp;scope&nbsp;of&nbsp;this&nbsp;thesis,&nbsp;it&nbsp;was&nbsp;aimed&nbsp;to&nbsp;develop&nbsp;effective&nbsp;mRNA&nbsp;vaccine&nbsp;transcripts&nbsp;against&nbsp;T.&nbsp;gondii&nbsp;and&nbsp;to&nbsp;determine&nbsp;the&nbsp;amount&nbsp;and&nbsp;immunogenicity&nbsp;of&nbsp;the&nbsp;recombinant&nbsp;protein&nbsp;which&nbsp;is&nbsp;produced&nbsp;with&nbsp;in&nbsp;vitro&nbsp;transcripts.&nbsp;For&nbsp;this&nbsp;purpose,&nbsp;firstly&nbsp;T.&nbsp;gondii&nbsp;GRA1&nbsp;protein&nbsp;was&nbsp;determined&nbsp;as&nbsp;the&nbsp;vaccine&nbsp;antigen&nbsp;and&nbsp;the&nbsp;solubility&nbsp;instability&nbsp;index,&nbsp;stability,&nbsp;half-life&nbsp;and&nbsp;antigenicity&nbsp;of&nbsp;GRA1&nbsp;protein&nbsp;were&nbsp;analyzed&nbsp;by&nbsp;in&nbsp;silico&nbsp;methods.&nbsp;Afterwards,&nbsp;the&nbsp;pEX-A258-GRA1&nbsp;vector&nbsp;carrying&nbsp;in&nbsp;vitro&nbsp;transcription&nbsp;(IVT)&nbsp;sequences&nbsp;including&nbsp;the&nbsp;GRA1&nbsp;gene&nbsp;was&nbsp;provided&nbsp;by&nbsp;service&nbsp;procurement.&nbsp;Following&nbsp;this&nbsp;step,&nbsp;the&nbsp;IVT&nbsp;sequence&nbsp;in&nbsp;the&nbsp;pEX-A258-GRA1&nbsp;vector&nbsp;was&nbsp;cloned&nbsp;into&nbsp;the&nbsp;pVAX1&nbsp;vector&nbsp;to&nbsp;enable&nbsp;the&nbsp;production&nbsp;of&nbsp;mRNA&nbsp;transcripts,&nbsp;and&nbsp;this&nbsp;vector&nbsp;was&nbsp;named&nbsp;pVAX1-GRA1.&nbsp;Four&nbsp;different&nbsp;types&nbsp;of&nbsp;mRNA&nbsp;vaccine&nbsp;transcripts&nbsp;were&nbsp;produced&nbsp;by&nbsp;IVT&nbsp;by&nbsp;linearizing&nbsp;pVAX1-GRA1.&nbsp;The&nbsp;mRNA&nbsp;vaccine&nbsp;transcripts&nbsp;were&nbsp;produced&nbsp;with&nbsp;a&nbsp;modified&nbsp;nucleoside,&nbsp;N1-methyl&nbsp;pseudouridine&nbsp;(m1Ψ&nbsp;+&nbsp;120&nbsp;bp)&nbsp;or&nbsp;wild-type&nbsp;uridine&nbsp;(uridine&nbsp;+&nbsp;120&nbsp;bp).&nbsp;In&nbsp;addition,&nbsp;by&nbsp;adding&nbsp;~150&nbsp;bp&nbsp;adenine&nbsp;bases&nbsp;to&nbsp;the&nbsp;end&nbsp;of&nbsp;these&nbsp;two&nbsp;mRNA&nbsp;vaccine&nbsp;transcripts,&nbsp;two&nbsp;more&nbsp;different&nbsp;ones&nbsp;(m1Ψ&nbsp;+&nbsp;120&nbsp;bp&nbsp;+&nbsp;poly-A&nbsp;tail&nbsp;added&nbsp;and&nbsp;uridine&nbsp;+&nbsp;120&nbsp;bp&nbsp;+&nbsp;poly-A&nbsp;tail&nbsp;added)&nbsp;were&nbsp;produced.&nbsp;IVT-produced&nbsp;mRNA&nbsp;vaccine&nbsp;transcripts&nbsp;were&nbsp;purified&nbsp;and&nbsp;then&nbsp;transfected&nbsp;into&nbsp;HEK293T&nbsp;cells,&nbsp;and&nbsp;the&nbsp;expression&nbsp;level&nbsp;and&nbsp;immunogenicity&nbsp;of&nbsp;the&nbsp;expressed&nbsp;recombinant&nbsp;GRA1&nbsp;protein&nbsp;were&nbsp;determined&nbsp;using&nbsp;IFAT,&nbsp;Western&nbsp;blot&nbsp;and&nbsp;ELISA.According&nbsp;to&nbsp;the&nbsp;results&nbsp;obtained,&nbsp;cell&nbsp;lysates&nbsp;were&nbsp;collected&nbsp;24&nbsp;hours&nbsp;after&nbsp;transfection&nbsp;and&nbsp;protein&nbsp;expression&nbsp;was&nbsp;examined&nbsp;by&nbsp;Western&nbsp;blot&nbsp;and&nbsp;it&nbsp;was&nbsp;determined&nbsp;that&nbsp;the&nbsp;highest&nbsp;expression&nbsp;was&nbsp;seen&nbsp;with&nbsp;the&nbsp;“uridine&nbsp;+&nbsp;120&nbsp;bp”&nbsp;mRNA&nbsp;vaccine&nbsp;transcripts,&nbsp;while&nbsp;the&nbsp;lowest&nbsp;protein&nbsp;expression&nbsp;was&nbsp;seen&nbsp;in&nbsp;cells&nbsp;transfected&nbsp;with&nbsp;the&nbsp;“m1Ψ&nbsp;+&nbsp;120&nbsp;bp&nbsp;+&nbsp;poly-A&nbsp;tail&nbsp;added”&nbsp;mRNA&nbsp;vaccine&nbsp;transcripts.&nbsp;This&nbsp;result&nbsp;was&nbsp;confirmed&nbsp;by&nbsp;IFAT&nbsp;and&nbsp;ELISA,&nbsp;and&nbsp;it&nbsp;was&nbsp;determined&nbsp;that&nbsp;“uridine&nbsp;+&nbsp;120&nbsp;bp”&nbsp;mRNA&nbsp;vaccine&nbsp;transcripts&nbsp;expressed&nbsp;more&nbsp;protein&nbsp;than&nbsp;“m1Ψ&nbsp;+&nbsp;120&nbsp;bp”&nbsp;mRNA&nbsp;vaccine&nbsp;transcripts.&nbsp;In&nbsp;addition,&nbsp;it&nbsp;was&nbsp;determined&nbsp;that&nbsp;adding&nbsp;a&nbsp;~150&nbsp;bp&nbsp;poly-A&nbsp;tail&nbsp;to&nbsp;mRNA&nbsp;vaccine&nbsp;transcripts&nbsp;did&nbsp;not&nbsp;increase&nbsp;protein&nbsp;expression&nbsp;levels.&nbsp;The&nbsp;immunogenicity&nbsp;of&nbsp;the&nbsp;recombinant&nbsp;GRA1&nbsp;protein&nbsp;expressed&nbsp;after&nbsp;transfection&nbsp;of&nbsp;4&nbsp;different&nbsp;mRNA&nbsp;vaccine&nbsp;transcripts&nbsp;produced&nbsp;by&nbsp;IVT&nbsp;into&nbsp;HEK293T&nbsp;cells&nbsp;was&nbsp;determined&nbsp;by&nbsp;Western&nbsp;blot&nbsp;and&nbsp;ELISA&nbsp;using&nbsp;T.&nbsp;gondii&nbsp;infected&nbsp;mouse&nbsp;sera.&nbsp;In&nbsp;conclusion,&nbsp;mRNA&nbsp;vaccine&nbsp;transcripts&nbsp;were&nbsp;successfully&nbsp;expressed&nbsp;recombinant&nbsp;GRA1&nbsp;protein&nbsp;in&nbsp;HEK293T&nbsp;cell&nbsp;line&nbsp;and&nbsp;the&nbsp;produced&nbsp;GRA1&nbsp;protein&nbsp;was&nbsp;determined&nbsp;to&nbsp;be&nbsp;immunogenic.&nbsp;Moreover,&nbsp;mRNA&nbsp;vaccine&nbsp;transcripts&nbsp;containing&nbsp;wild&nbsp;type&nbsp;uridine&nbsp;or&nbsp;m1Ψ&nbsp;could&nbsp;be&nbsp;used&nbsp;as&nbsp;potential&nbsp;vaccine&nbsp;candidates&nbsp;after&nbsp;encapsulation&nbsp;into&nbsp;lipid&nbsp;nanoparticles&nbsp;</p>

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