Optimization of Prime-Boost Vaccination Strategies Against Mouse-Adapted Ebolavirus in a Short-Term Protection Study

In nonhuman primates, complete protection against an Ebola virus (EBOV) challenge has previously beenachieved after a single injection with several vaccine platforms. However, long-term protection against EBOVafter a single immunization has not been demonstrated to this date. Interestingly, prime-boost regimens havedemonstrated longer protection against EBOV challenge, compared with single immunizations. Since prime-boost regimens have the potential to achieve long-term protection, determining optimal vectorcombinations iscrucial.However,testingprime-boostefficiencyinlong-termprotectionstudiesistimeconsumingandresourcedemanding. Here, we investigated the optimal prime-boost combination, using DNA, porcine-derived adeno-associated virus serotype 6 (AAV-po6), and human adenovirus serotype 5 (Ad5) vector, in a short-term protec-tion study in the mouse model of EBOV infection. In addition, we also investigated which immune parameterswere indicative of a strong boost. Each vaccine platform was titrated in mice to identify which dose (single im-munization) induced approximately 20% protection after challenge with a mouse-adapted EBOV. These doseswerethenusedtodeterminetheprotectionefficacyofvariousprime-boostcombinations,usingthesamemousemodel. In addition, humoral and cellular immune responses against EBOV glycoprotein were analyzed by anenzyme-linkedimmunosorbentassay,aneutralizingantibodyassay,andaninterferonγ–specificenzyme-linkedimmunospot assay. When DNA was used as a prime, Ad5 boost induced the best protection, which correlatedwith a higher cellular response. In contrast, when AAV-po6 or Ad5 were injected first, better protection wasachieved after DNA boost, and this correlated with a higher total glycoprotein-specific immunoglobulin Gtiter. Prime-boost regimens using independent vaccine platforms may provide a useful strategy to inducelong-term immune protection against filoviruses.Keywords. Ebola virus; prime-boost; vaccine; adenovirus; DNA; adeno-associated virus.Ebolavirus (EBOV) causes the most severe form ofhemorrhagicfeverknowntodate.Currentmanagementof EBOV-infected individuals mainly consists of sup-portive care, as no approved treatments or vaccinesagainst EBOV are commercially available [1, 2]. Thelack of approved therapeutics, high mortality rate, andease of transmission have prompted research on the de-velopment of both treatments and vaccines againstEBOV.Protection against EBOV challenge has been demon-strated in nonhuman primates (NHPs) after immuniza-tion with various vaccine platforms based on adenovirus(Ad) [3–5],vesicular stomatitis virus (VSV) [ 6],virus-likeparticles [7], human parainfluenza virus type 3 (HPIV3)[8],Newcastlediseasevirus[9],andreplication-competentrabies virus vector [10] encoding EBOV glycoprotein(GP). The efficacy of these platforms was mainly demon-strated28daysafterimmunization.However,lackoflong-term protection by these vaccine platforms would limittheir clinical use. Although protection against Marburgvirus challenge has been reported in NHPs approximately14 months after a single shot of VSV [ 11], to date, long-termprotectiveimmunityagainstEBOVhasonlybeenre-ported after a single dose of replication-competent rabiesvirus vector or after prime-boost regimens. After DNA/Ad5 or ChAd3/MVA prime-boost, NHPs were fully