GuideRead：Newly Approved Respiratory Syncytial Virus (RSV) Subunit andmRNAVaccines mainly provide protection by producing antibodies.80%Protection can effectively prevent diseases. Moreover, based on self-amplificationmRNA（saRNA) The alternative vaccine technology platform is expected to induce humoral and cellular immune responses, thereby serving as a preventiveRSVVaccine.

202411Month, Johnson & Johnson vaccine R&D team inNature Communications（IF:14.7) Published on“A self-amplifying RNA RSV prefusion-F vaccine elicits potent immunity in pre-exposed and naïve non-human primates”, EvaluationLNPEncapsulatedsaRNAVaccine (SMARRT.RSV.preF) The immunogenicity of the vaccine, when administered intramuscularly innaïveMice and non-human primates (NHP) Induction in ChinaRSVNeutralizing Antibodies andTh1Biased cellular response,RSVPre-exposureNHPSingle-dose vaccination triggers a dose-dependent memory humoral immune response. This is also the reason why Johnson & Johnson terminated the adenovirus vector.RSVAfter the development of the vaccine, targetingRSVThe announcement of another new vaccine technology.

01

Background Introduction

RSVSingle-stranded negative chainRNAVirus, with spikes on the double-layer lipid envelope, namelyGAndFProtein.GHas an adsorption effect on host cells,FFusion protein.RSVIt is the most important pathogen causing lower respiratory tract infections in infants and young children worldwide, particularly viral pneumonia and bronchiolitis in infants. However, poor induction of neutralizing antibodies,Th2BiasedCD4⁺ TCellular Response,I/IITypeIFNReduced response and based on animal studiesRSVFactors such as increased eosinophils in the lungs after infection make it effectiveRSVThe development of vaccines has become complicated.

Newborn MiceRSVThe infection model shows that during the initial infectionI/IITypeIFNThe reaction led to a reduction in lung pathology upon reinfection;Th1BiasedRSV.FThe vaccine has been proven to overcome in animal modelsTh2Bias, Promote Neutralizing Antibodies andTInduction of cells. Moreover, studies have shownRSVSpecific FunctionTThe reduction in cells is associated with the severity of illness in the elderly. Therefore,Effectively trigger humoral immunity andTh1Biased Cellular Immune ResponseRSVVaccines may be crucial for protecting these vulnerable populations.。

02

Design and In Vitro Characterization of the SMARRT.RSV.preF Vaccine

InVEEV TC83The replicon sequence of the attenuated strain as thesaRNAThe backbone of the vaccine, will expressRSV A2Full-length strain, membrane-anchored, stableRSVBefore FusionFProtein (pre-F) after the sequence is inserted downstream of the backbone subgenome promoter, using a formulation containing ionizable lipidsALC-0315, Cholesterol,DSPCAndDMG-PEG2000TheLNPEncapsulated to obtain vaccine finished productSMARRT.RSV.preF. In addition, to avoid the impact of the host's innate response on replicon translation, innSP-1Upstream insertion of a structure known as the downstream loop (DLP) ofRNAMotif (fromSindbisVirus).SMARRT.RSV.preFThe average hydrodynamic particle size is75±5nm，PDI＜0.2. Use anti-RSV.FSpecificitymAbDuring vaccination24hLaterBHKTotal expression and surface expression of membrane-anchored proteins confirmed by flow cytometry in cellsRSV.FThe proportion of cells.

Figure1 SMARRT.RSV.preFDesign and In Vitro Activity

03

Immunogenicity of SMARRT.RSV.preF in Mouse Models

8Only6-8Week-old femaleBALB/cMice were injected intramuscularly, respectively.0.1、1And10mcg SMARRT.RSV.preF（100μL/Only, one on each hind leg.50μL），28dThe second dose was administered later, while the positive control received a single dose.10¹⁰IndividualAd26.RSV.preFVirus particles. Single-dose vaccinationSMARRT.RSV.preFAfterward, in the mouse serumRSV-CL57Neutralizing Antibody (VNA) andRSV-preFSpecificityIgGThe antibodies increased in a dose-dependent manner, and the serum antibodies significantly increased after the secondary immunization.

Virus Neutralization Test（naïveAndRSVPre-exposed mice and cynomolgus monkeysNHP）：Serially dilute the heat-inactivated animal serum samples and25000pfuFirefly Luciferase (FFL) MarkedRSV-CL57After mixing, at room temperature (RT) Incubation1h, add to each well5000IndividualA549Cell (Infectious Plural:5), place the board in37℃、10%CO₂Incubation20hLater AdditionNeoliteSubstrate, usedEnvision®The plate reader measures the luminescence signal.

ELISATest(Determination of Mice and Cynomolgus MonkeysNHPIn serumRSV preFSpecificityIgGAntibody andNHPIn Nasal SwabRSV preFSpecificityIgATiter)：

Coated with0.5μg/mLStreptavidin96Half-area Plate with Holes4℃Incubate overnight. Using0.05% Tween-20ThePBSWash the bore with1%CaseinPBSBufferRTClosed1hAfter washing, the biotinylated2μg/mL RSV preFProtein added to the wellRTIncubation1hAfter washing, add the heat-inactivated serum samples and standards to the wells.RTIncubation1h. UseHRPLabeled Anti-MouseIgG（1：5000) or anti-monkeyIgA（1：5000) DetectionRSV preFSpecific antibody.

To evaluate cellular responses, useRSV.FPeptide Library Stimulation Vaccination2Dose10mcg SMARRT.RSV.preFMouse spleen cells (56Day separation),ICSResults ShowSMARRT.RSV.preFInductionIFNγ⁺AndTNFα⁺Single Functionality andIFNγ⁺TNFα⁺IL2⁺AndIFNγ⁺TNFα⁺MultifunctionalRSV.FSpecificityCD8⁺ TCells. And induce low-frequency single-functionCD4⁺ TCells andIFNγ⁺TNFα⁺IL2⁺、IFNγ⁺TNFα⁺、TNFα⁺IL2α⁺Double-positive cells.In summary,SMARRT.RSV.preFIt is immunogenic in mice after a single immunization, and the response can be further increased after a second immunization.

Measurement Method:Used byRSV.A2、Hamster Anti-MouseCD28（1:500) and rat anti-mouseCD49d（1:500) ofFProtein Peptide Library Stimulates Spleen Cells1hLater AdditionBD GolgiPlug™. Placed in37℃、5% CO₂Lower Incubation4hLater in4℃And5% CO₂Incubate overnight. Then use anti-mouseCD16/CD32Antibody (1:50) BlockadeFcReceptor, further with anti-CD3-FITC（1:400，Clone142-2C11）、CD4-PerCpCy5.5（1:400，CloneRM4-5) andCD8-APC.H7（1:75，Clone53-6.7) Stain the cells. WithBD Cytofix/Cytoperm™Perform permeabilization, and useIFNγ-PE（1:200，CloneXMG1.2）、TNFα-PE.Cy7（1:200，CloneMP6-XT22) andIL2-APC（1:300，CloneJES6-5H4) Antibody for intracellular staining, and finally analyze the proportion of positive cells by flow cytometry.

Figure2 SMARRT.RSV.preFInduction of Humoral and Cellular Responses in Mice

04

SMARRT.RSV.preF Induces Humoral Immunity in NHP

InnaïveAndRSVPre-exposureNHPMid-term EvaluationSMARRT.RSV.preFImmunogenicity.12Only6-7Age of the Cynomolgus MonkeyNHPInfection via Nasal and Endotracheal Routes10⁶PFU RSV.A2OrRSV_Memphis37(Nasal0.1mLEndotracheal1mL, totaling1.1mL), intramuscular injection after three months1Or10mcg SMARRT.RSV.preFOr on the0Weekly Vaccination50mcgReorganizationRSV.preFProtein (PRPM）；naïveControl Group (n=4) In the0Weekly Vaccination10mcg SMARRT.RSV.preF. Results inRSVPre-exposureNHPIn China,PRPM、1Or10 mcg SMARRT.RSV.preFGroup D2Zhou's MemoryVNAReaction andIgGTiter comparison0Significantly improved on a weekly basis, and after vaccination3The average level was significantly higher than before vaccination after several months.naïve NHPIn the group,VNAReaction andIgGThe titer also increased significantly, but was clearly weaker than the same dose ofRSVPre-exposure group.

Research shows,RSVSpecific MucosaIgAAntibody and Human &NHPis related to the protective effect. Therefore, the measurement was taken on the8Nasal swabs collected by Zhou containedRSV.preFSpecificityIgA. AndnaïveGroup (1/4) Compared with vaccination10mcg SMARRT.RSV.preFAll ofRSVPre-exposure animals and3/4Vaccination1mcg SMARRT.RSV.preFin animals with detectableIgAAntibody.In summary,SMARRT.RSV.preFInRSVPre-exposureNHPInducing memory serological reactions in China, accompanied by detectable levels in the nasal cavityRSV.preF IgAAntibody.

Figure3 SMARRT.RSV.preFInNHPInduction of Humoral Immunity in China

05

SMARRT.RSV.preF Induces Cellular Immunity in NHP

ThroughIFNγ ELISpotMeasurement from after vaccine immunizationNHPIsolated fromPBMCThe antigen-specific cellular response, counted as per millionPBMCCellularIFN-γFormation Unit (SFU）。RSVPre-exposureNHPIn China,10mcg SMARRT.RSV.preFGroup'sELISpotReaction compared to1mcgHigher and more consistent group peaks,PRPMThe group did not increase.RSV.FSpecificityIFNγ ELISpotReaction.naïveGroup D4Weekly induction is weaker than the same dose.RSVPre-exposure Groupde-novoReaction.

Whole BodyRSV.FSpecific MultifunctionalTCells are associated with a low reinfection rate in humans.ELISpotConsistent response,SMARRT.RSV.preFAfter immunization,RSVPre-exposureNHPIn the4Week compared to baseline,RSV-FSpecific MemoryCD4⁺AndCD8⁺ TCell proliferation,PRPMThe group did not increase.naïveIn the group,10mcg SMARRT.RSV.preFGroup MemoryCD4⁺ TSlight increase in cells, toCD8⁺ THas less impact on cells, and when the same dose is administeredRSVMemory of the Pre-exposure GroupCD4⁺ TCells are relativelynaïveGroup Increase3Times.

Figure4 IFNγ ELISpotAndRSV.FSpecific MultifunctionalTCell Assay

AdoptSPICEDetermination of the4Vaccine-induced weeklyTCell多功能性。Protein vaccination后仅1/4MemoryCD4⁺AndCD8⁺ TCells exhibit multifunctionality, and the predominant phenotype of single-function cells isTNF-αPositive.1mcgOr10mcg SMARRT.RSV.preFGroup'sRSVPre-exposureNHPShow Ultra50%MultifunctionalCD4⁺AndCD8⁺ TCell, Among themIFNγ⁺TNFα⁺IL2⁺Triple-Positive MemoryCD4⁺ TThe number of cells isnaïveGroup's10Times;CD107a⁺IFNγ⁺TNFα⁺IL2⁺、CD107a⁺IFNγ⁺TNFα⁺AndCD107a⁺IFNγ⁺Cell OccupancyRSV.FSpecificityCD8⁺ TCellular50%, and these multifunctionalTThe most secreted in cells isCD107aAndIFNγ，naïveThe group mainly inducesCD107aAndTNFαSingle-function MemoryCD8⁺ TCell.

In summary, inRSVPre-exposureNHPIn China, single doseSMARRT.RSV.preFCompared toRSVProtein vaccines induce higher levels, higher quality multifunctional memoryTCell.

 

Figure5 SPICEMeasurementTCellular Pluripotency

06

SMARRT.RSV.preF Induces Inflammatory Cytokines Involved in Immune Cell Chemotaxis

The early innate immune response after vaccination will influence the adaptive immune response. Measure before and after immunization.24hIn the serum45Analyte (Inflammation-Related Cytokines/Chemokine) levels.PRPMImmune did not cause any change in analyte levels compared to baseline.1 mcgAnd10 mcg SMARRT.RSV.preFGroup Immunity24hLater, there are respectively9And23The level of an analyte changes, among which pro-inflammatory analytes (such asIL6、TNFα、IL18、IL15、VEGFA) and participate in immune cell trafficking/Regulated analytes (such asCXCL9、CXCL10、CXCL11、CSF1、Flt3LG、CCL-3、4、8、11And19) Level上调；EGFRLigandEGFAndTGFαand plays an important role in cell migration and angiogenesisMMP-1AndMMP-12Horizontal downward adjustment. The results of inter-analyte correlation analysis showed includingIL15、CCL4、CXCL10AndTNFαA series of cytokines, including/Chemokines in RegulationSMARRT.RSV.preFSynergistic effects in induced innate responses. Enrichment analysis revealed that the analyte affects interferons (IAndIIType) Signal pathway-mediated inflammation-related typical pathway.

In summary,SMARRT.RSV.preFInduction of multipotent cytokines primarily involved in inflammation and immune cell chemotaxis/Dose-dependent increase in chemokines.

 

Figure6 SMARRT.RSV.preFInduced Serum Chemokines/Cytokine Analysis

07

Conclusion

This study is the first to describe based onsaRNATheRSVVaccine innaïveAndRSVInfectedNHPAnd it has immunogenicity in China, and a single dose has been provenSMARRT.RSV.preFIn mice andnaïve/RSVPre-infectedNHPInduce effective humoral and cellular responses in China. InRSVIn the pre-exposure environment, compared toRSV.FProtein vaccine,SMARRT.RSV.preFThe ability to induce humoral responses was comparable, but the intensity and quality of cellular responses were significantly enhanced.

In summary,saRNAPlatform innaïveAnd induce at relatively low doses in pre-exposure individualsTh1The inherent ability to respond and enhance immune responses makes it suitable for both children and adults.RSVThe ideal platform for vaccines.

[1] Vijayan A, Vogels R, Groppo R, Jin Y, Khan S, Van Kampen M, Jorritsma S, Boedhoe S, Baert M, van Diepen H, Kuipers H, Serroyen J, Del Valle JR, Broman A, Nguyen L, Ray S, Jarai B, Arora J, Lifton M, Mildenberg B, Morton G, Santra S, Grossman TR, Schuitemaker H, Custers J, Zahn R. A self-amplifying RNA RSV prefusion-F vaccine elicits potent immunity in pre-exposed and naïve non-human primates. Nat Commun. 2024 Nov 14;15(1):9884. doi: 10.1038/s41467-024-54289-9.

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