Demonstration of Immunogenicity of a DNA Vaccine Model Developed Against the Omicron Variant of SARS-CoV-2, the Causative Agent of COVID-19

Project no. 32657 · Status: Kapanmış (Yayın bekleniyor) · 2025

Summary

<p>The&nbsp;causative&nbsp;agent&nbsp;of&nbsp;coronavirus&nbsp;disease&nbsp;19&nbsp;(COVID-19),&nbsp;Severe&nbsp;Acute&nbsp;Respiratory&nbsp;Syndrome&nbsp;Coronavirus&nbsp;2&nbsp;(SARS-CoV-2),&nbsp;emerged&nbsp;in&nbsp;Wuhan,&nbsp;China,&nbsp;in&nbsp;late&nbsp;2019&nbsp;and&nbsp;quickly&nbsp;led&nbsp;to&nbsp;a&nbsp;pandemic,&nbsp;turning&nbsp;into&nbsp;a&nbsp;global&nbsp;public&nbsp;health&nbsp;crisis.&nbsp;This&nbsp;virus,&nbsp;which&nbsp;has&nbsp;a&nbsp;positive-sense&nbsp;single-stranded&nbsp;RNA&nbsp;(+ssRNA)&nbsp;genome,&nbsp;has&nbsp;continued&nbsp;to&nbsp;spread&nbsp;by&nbsp;forming&nbsp;different&nbsp;variants&nbsp;due&nbsp;to&nbsp;mutations&nbsp;in&nbsp;its&nbsp;genetic&nbsp;structure&nbsp;over&nbsp;time.&nbsp;Most&nbsp;recently,&nbsp;the&nbsp;KP.3.1.1&nbsp;variant,&nbsp;a&nbsp;sublineage&nbsp;of&nbsp;Omicron&nbsp;BA.2.86,&nbsp;has&nbsp;attracted&nbsp;attention&nbsp;due&nbsp;to&nbsp;a&nbsp;serine&nbsp;31&nbsp;deletion&nbsp;and&nbsp;F456L,&nbsp;Q493E,&nbsp;and&nbsp;V1104L&nbsp;mutations&nbsp;in&nbsp;the&nbsp;Spike&nbsp;(S)&nbsp;protein.&nbsp;These&nbsp;mutations&nbsp;increase&nbsp;the&nbsp;virus's&nbsp;ability&nbsp;to&nbsp;evade&nbsp;the&nbsp;immune&nbsp;system&nbsp;and&nbsp;enhance&nbsp;the&nbsp;transmissibility&nbsp;of&nbsp;the&nbsp;disease.&nbsp;The&nbsp;observed&nbsp;decrease&nbsp;in&nbsp;the&nbsp;effectiveness&nbsp;of&nbsp;current&nbsp;vaccines&nbsp;against&nbsp;these&nbsp;newly&nbsp;emerging&nbsp;variants&nbsp;highlights&nbsp;the&nbsp;importance&nbsp;of&nbsp;more&nbsp;flexible&nbsp;and&nbsp;rapidly&nbsp;producible&nbsp;alternative&nbsp;vaccine&nbsp;platforms.&nbsp;In&nbsp;this&nbsp;context,&nbsp;DNA&nbsp;vaccines&nbsp;have&nbsp;emerged&nbsp;as&nbsp;promising&nbsp;biotechnological&nbsp;vaccine&nbsp;candidates&nbsp;due&nbsp;to&nbsp;their&nbsp;high&nbsp;structural&nbsp;stability,&nbsp;rapid&nbsp;production&nbsp;processes,&nbsp;and&nbsp;ability&nbsp;to&nbsp;trigger&nbsp;both&nbsp;humoral&nbsp;and&nbsp;cellular&nbsp;immune&nbsp;responses.</p><p>In&nbsp;this&nbsp;study,&nbsp;a&nbsp;DNA&nbsp;vaccine&nbsp;targeting&nbsp;the&nbsp;Spike&nbsp;protein&nbsp;specific&nbsp;to&nbsp;the&nbsp;KP.3.1.1&nbsp;variant&nbsp;and&nbsp;optimized&nbsp;for&nbsp;human&nbsp;codons&nbsp;was&nbsp;designed.&nbsp;To&nbsp;ensure&nbsp;prefusion&nbsp;stability,&nbsp;six&nbsp;proline&nbsp;substitutions&nbsp;were&nbsp;implemented;&nbsp;an&nbsp;IgE&nbsp;signal&nbsp;peptide&nbsp;and&nbsp;Kozak&nbsp;sequence&nbsp;were&nbsp;added;&nbsp;the&nbsp;gene&nbsp;was&nbsp;cloned&nbsp;into&nbsp;the&nbsp;FDA-approved&nbsp;pVAX1&nbsp;vector.&nbsp;The&nbsp;resulting&nbsp;pVAX1-KP.3.1.1-Spike&nbsp;plasmid&nbsp;was&nbsp;purified&nbsp;using&nbsp;an&nbsp;endotoxin-reducing&nbsp;kit&nbsp;and&nbsp;transfected&nbsp;into&nbsp;HEK293T&nbsp;cells,&nbsp;with&nbsp;expression&nbsp;confirmed&nbsp;by&nbsp;Western&nbsp;blot,&nbsp;IFAT,&nbsp;and&nbsp;RT-qPCR&nbsp;analyses.&nbsp;For&nbsp;in&nbsp;vivo&nbsp;studies&nbsp;of&nbsp;the&nbsp;vaccine,&nbsp;BALB/c&nbsp;mice&nbsp;were&nbsp;vaccinated&nbsp;with&nbsp;the&nbsp;pVAX1-KP.3.1.1-Spike&nbsp;vaccine&nbsp;or&nbsp;empty&nbsp;pVAX1&nbsp;vector&nbsp;via&nbsp;both&nbsp;IM&nbsp;and&nbsp;ID+EP&nbsp;methods&nbsp;on&nbsp;days&nbsp;0,&nbsp;14,&nbsp;and&nbsp;56.&nbsp;The&nbsp;immune&nbsp;responses&nbsp;elicited&nbsp;by&nbsp;vaccination&nbsp;were&nbsp;analyzed&nbsp;by&nbsp;flow&nbsp;cytometry&nbsp;for&nbsp;IFN-γ&nbsp;and&nbsp;IL-4-secreting&nbsp;T&nbsp;cells&nbsp;in&nbsp;splenocytes&nbsp;isolated&nbsp;after&nbsp;the&nbsp;final&nbsp;vaccination;&nbsp;extracellular&nbsp;cytokine&nbsp;levels&nbsp;were&nbsp;measured&nbsp;by&nbsp;ELISA.&nbsp;The&nbsp;humoral&nbsp;response&nbsp;was&nbsp;evaluated&nbsp;by&nbsp;the&nbsp;presence&nbsp;of&nbsp;antigen-specific&nbsp;IgG&nbsp;antibodies&nbsp;in&nbsp;serum&nbsp;samples&nbsp;using&nbsp;Western&nbsp;blot,&nbsp;ELISA,&nbsp;and&nbsp;Surrogate&nbsp;Neutralization&nbsp;test.&nbsp;</p><p>pVAX1-KP.3.1.1-Spike&nbsp;vaccination&nbsp;in&nbsp;the&nbsp;IM&nbsp;and&nbsp;ID+EP&nbsp;groups&nbsp;showed&nbsp;an&nbsp;increase&nbsp;in&nbsp;IFN-γ&nbsp;production&nbsp;by&nbsp;CD8+&nbsp;and&nbsp;CD4+&nbsp;T&nbsp;cells&nbsp;compared&nbsp;to&nbsp;control&nbsp;groups&nbsp;(CD8+:&nbsp;IM&nbsp;≈2-fold,&nbsp;p=0.2247;&nbsp;ID+EP&nbsp;≈2.6-fold,&nbsp;p=0.0445;&nbsp;CD4+:&nbsp;IM&nbsp;≈5.8-fold,&nbsp;p=0.0372;&nbsp;ID+EP&nbsp;≈1.8-fold,&nbsp;p=0.0217).&nbsp;No&nbsp;significant&nbsp;difference&nbsp;in&nbsp;IL-4&nbsp;secretion&nbsp;was&nbsp;observed&nbsp;among&nbsp;the&nbsp;groups&nbsp;in&nbsp;T&nbsp;cells.&nbsp;ELISA&nbsp;analyses&nbsp;performed&nbsp;on&nbsp;splenocyte&nbsp;supernatants&nbsp;showed&nbsp;that&nbsp;IFN-γ&nbsp;levels&nbsp;were&nbsp;significantly&nbsp;elevated&nbsp;in&nbsp;both&nbsp;vaccine&nbsp;groups&nbsp;compared&nbsp;to&nbsp;the&nbsp;control&nbsp;group&nbsp;(IM:&nbsp;p=0.0407;&nbsp;ID+EP:&nbsp;p=0.0188),&nbsp;while&nbsp;IL-4&nbsp;levels&nbsp;remained&nbsp;low.</p><p>The&nbsp;pVAX1-KP.3.1.1-Spike&nbsp;DNA&nbsp;vaccine&nbsp;highly&nbsp;expressed&nbsp;the&nbsp;KP.3.1.1&nbsp;Spike&nbsp;protein&nbsp;in&nbsp;HEK293T&nbsp;cells.&nbsp;Serum&nbsp;samples&nbsp;collected&nbsp;on&nbsp;day&nbsp;70&nbsp;from&nbsp;BALB/c&nbsp;mice&nbsp;vaccinated&nbsp;with&nbsp;the&nbsp;pVAX1-KP.3.1.1-Spike&nbsp;vaccine&nbsp;showed&nbsp;significant&nbsp;increases&nbsp;in&nbsp;both&nbsp;total&nbsp;IgG&nbsp;and&nbsp;IgG&nbsp;subclasses&nbsp;against&nbsp;the&nbsp;SARS-CoV-2&nbsp;KP.3.1.1&nbsp;RBD&nbsp;protein.&nbsp;The&nbsp;total&nbsp;IgG&nbsp;response&nbsp;in&nbsp;the&nbsp;IM&nbsp;group&nbsp;was&nbsp;approximately&nbsp;11-fold&nbsp;higher&nbsp;than&nbsp;in&nbsp;the&nbsp;control&nbsp;group&nbsp;(P=0.0025),&nbsp;while&nbsp;the&nbsp;ID+EP&nbsp;group&nbsp;showed&nbsp;an&nbsp;approximately&nbsp;12-fold&nbsp;increase&nbsp;(P=0.0004).&nbsp;Only&nbsp;the&nbsp;ID+EP&nbsp;group&nbsp;showed&nbsp;a&nbsp;significant&nbsp;increase&nbsp;in&nbsp;IgG1&nbsp;response&nbsp;compared&nbsp;to&nbsp;the&nbsp;control&nbsp;group&nbsp;(P=0.0007),&nbsp;and&nbsp;this&nbsp;group&nbsp;also&nbsp;produced&nbsp;a&nbsp;higher&nbsp;IgG1&nbsp;response&nbsp;than&nbsp;the&nbsp;IM&nbsp;group&nbsp;(P=0.0111).&nbsp;IgG2a&nbsp;levels&nbsp;were&nbsp;significantly&nbsp;increased&nbsp;in&nbsp;both&nbsp;vaccine&nbsp;groups&nbsp;compared&nbsp;to&nbsp;the&nbsp;control&nbsp;groups&nbsp;(P&lt;0.001),&nbsp;but&nbsp;there&nbsp;was&nbsp;no&nbsp;statistical&nbsp;difference&nbsp;between&nbsp;the&nbsp;vaccine&nbsp;groups.</p><p>In&nbsp;conclusion,&nbsp;the&nbsp;DNA&nbsp;vaccine&nbsp;targeting&nbsp;the&nbsp;KP.3.1.1&nbsp;variant&nbsp;was&nbsp;found&nbsp;to&nbsp;effectively&nbsp;induce&nbsp;both&nbsp;cellular&nbsp;and&nbsp;humoral&nbsp;immune&nbsp;responses&nbsp;and&nbsp;may&nbsp;represent&nbsp;an&nbsp;alternative&nbsp;and&nbsp;promising&nbsp;vaccine&nbsp;strategy&nbsp;that&nbsp;can&nbsp;be&nbsp;rapidly&nbsp;produced&nbsp;against&nbsp;new&nbsp;SARS-CoV-2&nbsp;variants.</p>

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