Virulent Pathogen Vaccines Based On Recombinant Adenoviral Vector

Ebola virus and Bacillus anthracis are Class A biological warfare agents which canbe used for bioterrorism. In this regard, developing effective vaccines that can inducea rapid and sustained immune response is a priority for the prevention ofbioterrorism-associated Ebola and anthrax infections. Here, we developedrecombinant replication-deficient adenovirus serotype5based vaccines expressing theSudan or Zaire Ebola virus glycoprotein （Ad5-GPS, Ad5-GPZ） or the humanizedprotective antigen （Ad5-PAopt）, and evaluated their immunogenicity in mouse and ratmodels.In order to develop a Ebola virus vaccine, the most toxic subtypes of the Ebolavirus, Sudan and Zaire Ebola virus, were chose as the object. The gene of theenvelope glycoprotein （GP） of Sudan and Zaire Ebola virus were synthesized andcloned into the AdMax adenovirus shuttle plasmid pDC316, and co-transfected intoHEK293cells with the backbone plasmid pBHGlox_E1,3Cre. Site-specificrecombination occurred and the recombinant adenoviral vector-based vaccineexpressing the GP of Ebola virus was packaged. They were amplified and purified toget a certain amount for immunological evaluation.The cellular immune response plays an important role in mediating vaccine-inducedprotective immunity for Ebola virus; besides, the Cytotoxin T lymphocyte （CTL）epitope is the basis for evaluating the cellular immune response of the vaccine. Inorder to identify the CTL epitopes suited for BALB/c mice, Computer-assistedalgorithms were used to predict H-2^d-specific T cell epitopes in Sudan or Zaire Ebolavirus GP. The most likely CTL epitope were chose and synthesized for furtherevaluation. Enzyme-linked immunospot assays （ELISPOT） and intracellular cytokinestaining （ICS） showed that the peptides RF-9（Sudan Ebola virus）, GF-9and LV-9（Zaire Ebola virus） could stimulate splenoctyes in immunized mice to produce largeamounts of interferon-gamma （IFN-γ）. LV-9is specific to H-2Kdand had beenidentified previously; GF-9represented a novel epitope which specific to H-2Ld; RF-9 represents the first H-2^d-restricted peptide described in Sudan Ebola virus GP.Furthermore, GF-9was more efficient at inducing IFN-γ secretion than LV-9,suggesting that GF-9represented a more sensitive epitope for the detection of IFN-γ.Using the same strategy, we successfully identified a first HLA-A*0201specific CTLepitope, CV-9, in Sudan Ebola virus GP. The identification of these epitopes shouldfacilitate the evaluation of vaccines based on the Ebola virus GP.BALB/c mice were used for evaluating the immunogenicity of the recombinantadenovirus-based Ebola virus vaccine. ELISPOT assay and ICS showed that bothAd5-GPS and Ad5-GPZ can induce rapid and robust cellular immune response. Forthe humoral immune response of the vaccine, mice inoculated with those vaccinecandidates could produce high level of serum antibody titers and it could sustainnearly one year. We then immunized mice with the mixed Sudan and Zaire Ebolavirus vaccine, and a strong cellular immune response both for Sudan and Zaire Ebolavirus GP was detected. This result largely demonstrates that Ad5-GPS and Ad5-GPZcan be used simultaneously as a mix vaccine. At the same time, the IFN-γ positiveCD8cells were gated for detecting the expression of CD107and the secretion of TNFand IL-2. The result showed that about40%of IFN-γ positive CD8cells were IFN-γsingle positive cells; however, more than5%IFN-γ, TNF, IL-2and CD107tetra-positive cells were detected and the percentage of IFN-γ, TNF and IL-2tri-positive cells was greater than10%. This result suggested that a large proportion ofthe vaccine-induced effector T cells are multifunctional T cells. By comparing theimmune response of Ad5-GPZ delivery from different routes, the results showed that,intramuscular injection （IM） of Ad5-GPZ can induce a strong cellular immuneresponse, while intranasal delivery （IN） of the vaccine can produce a higher serumantibody titer. Although the secretion of IFN-γ couldn’t be detected in the splenocytesof those IN immunized mice, a relatively high level of secret IgA （sIgA） antibody inthe lung lavage was detected in the IN but not IM immunized mice. We nextinvestigated the immune response in mice vaccination with Ad5-GPZ in combinationof different delivery route, and found that intramuscular inoculation followed byintranasal vaccination could not only induce a robust cellular immune response butalso stimulate strong serum IgG antibody and lung lavage sIgA antibody titers. Finally,the codons of the secreted GP （sGP） of Zaire Ebola virus were humanized to preparethe condon optimized recombinant adenoviral-based Zaire Ebola virus vaccine（Ad5-sGPZopt）. The immunological evaluation of Ad5-sGPZopt showed that, although the cellular immune response of Ad5-sGPZopt was equal to Ad5-GPZ, asignificant higher of the serum antibody titers was observed in those Ad5-sGPZoptmice. At the same time, the Ad5-sGPZopt immunized mice had a significant earlier intime to produce anti-Ebola GP antibodies.For the anthrax vaccine, the codons of the anthrax protective antigen （PA） genewere optimized for expression in human cells, and the expression level of PAsignificantly increased after codon optimization in293T cells. In silico methodstogether with in vivo/in vitro validation had used for identification of CTL epitopes inPA, and a H-2^d-specific CTL epitope, NA-9，as well as two HLA-A*0201-specificCTL epitpoes, AV-9and FI-9, were discovered. A single intramuscular injection ofAd5-PAopt resulted in rapid and robust cellular immune response. For the humoralimmune response of the vaccine, both intramuscular and intranasal inoculation couldinduce strong and sustained serum anti-PA antibody and toxin neutralizing antibodyresponses. By detecting the subclasses of anti-PA specific antibodies, the resultsshowed that the main antibody subclass of intranasally immunized mice was IgG1,while intramuscularly immunized mice produced higher IgG2a antibody titers.Anthrax lethal toxin-sensitive Fisher344rats were used for anthrax lethal toxinchallenge; rats intramuscularly inoculated with a single dose of108infectious units ofAd5-PAopt achieved100%protection from challenge with10LD₅₀of anthrax lethaltoxin7days after vaccination, largely supporting its feasibility for use in emergencysituations.The potential pre-existing anti-vector immunity （PEI） must be considered indeveloping Ad5-based vaccines. Here, PEI to Ad5vector was induced byintramuscularly or intranasally inoculation mice with Ad5-EGFP, and the immuneresponse of Ad5-PAopt in the present of PEI were evaluated by following vaccinatedthose mice with Ad5-PAopt. The results showed that intranasal inoculation ofAd5-PAopt could bypass the anti-Ad5PEI initiated not only from the intramuscularroute but also from the intranasal route. The intramuscular originate PEI significantlyaffected the following intramuscular vaccination; however, the intranasal originatePEI only partially affected the immune responses of intramuscular Ad5-PAoptimmunized mice. In the present of intranasal originate PEI to Ad5,100%protectionwas achieved15days after intramuscular Ad5-PAopt injection in Fisher344rats. Asnatural Ad5infection often occurs via the mucosal route, those results largelyilluminated that intramuscular inoculation with Ad5-PAopt can overcome the negative effects of immunity induced by prior adenovirus infection and represents an efficientapproach for protection against emerging anthrax.In summary, the recombinant adenoviral vector-based Ebola virus and anthraxvaccine packaged here can induce rapid, robust and sustained both cellular andhumoral immune response, suggesting their feasibility for use in emergencysituations.