Attenuated human parainfluenza virus type 1 expressing Ebola virus glycoprotein GP administered intranasally is immunogenic in African green monkeys

The recent 2014-2016 Ebola virus (EBOV) outbreak prompted increased efforts to 20 develop vaccines against EBOV disease. We describe the development and preclinical evaluation 21 of an attenuated recombinant human parainfluenza virus type 1 (rHPIV1) expressing the 22 membrane-anchored form of EBOV glycoprotein GP, as an intranasal (IN) EBOV vaccine. GP 23 was codon optimized and expressed either as a full-length protein or an engineered chimeric form 24 in which its transmembrane and cytoplasmic tail (TMCT) domains were substituted with those of 25 the HPIV1 F protein in an effort to enhance packaging into the vector particle and 26 immunogenicity. GP was inserted either preceding the N gene (pre-N) or between the N and P genes (N-P) of rHPIV1 bearing a stabilized attenuating mutation in the P/C gene (C∆170 27 ). The 28 constructs grew to high titers and efficiently and stably expressed GP. Viruses were attenuated, 29 replicating at low titers over several days, in the respiratory tract of African green monkeys 30 (AGMs). Two doses of candidates expressing GP from the pre-N position elicited higher GP 31 neutralizing serum antibody titers than the N-P viruses, and unmodified GP induced higher levels 32 than its TMCT counterpart. Unmodified EBOV GP was packaged into the HPIV1 particle, and 33 the TMCT modification did not increase packaging or immunogenicity but rather reduced the 34 stability of GP expression during in vivo replication. In conclusion, we identified an attenuated 35 and immunogenic IN vaccine candidate expressing GP from the pre-N position. It is expected to 36 be well-tolerated in humans and is available for clinical evaluation.