Immune Protection Of Cytomegalovirus IE-1 Subunit Vaccine And DNA Vaccine

Human cytomegalovirus(HCMV)causes serious HCMV-related diseases in immunocompromised people,including newborns,AIDS patients,and organ transplant recipients.Vaccination is the most effective way to control its infection.Due to generally unsatisfactory efficacy of the candidate vaccines,no vaccine against HCMV has been licensed till now.It’s really worth knowing which of the antigens from HCMV are able to offer anti-infection protection and what immune strategies should be used.In this paper,we focused on MCMV IE-1 antigen,which has a homolog in HCMV,and explored the immune protection of IE-1 subunit vaccine and DNA vaccine against lethal virus challenge in mouse model.The following aspects are discussed: 1)Immune responses and protective effects in mice induced by prokaryotically expressed recombinant IE-1(rIE-1),and difference in immune protection between intraperitoneal(i.p.)immunization and intranasal(i.n.)immunization with the protein;2)Immune responses and protective effects in mice induced by combined immunization of IE-1 DNA and subunit vaccine.3)Immune protection in mice provided by five DNA vaccines expressing different antigens including IE-1.The paper consists of five parts:1.Introduction In this part,HCMV infection,research progress on the virus vaccines,functions of main genes and their proteins,as well as the potential protection in human offered by IE-1 were summarized.2.Prokaryotic expression of IE-1 The MCMV IE-1 gene was cloned and inserted into prokaryotic expression plasmid p ET28 a.The target protein was produced in E.coli.cells under IPTG induction and was purified.The purification conditions were optimized and the final purified protein was confirmed as rIE-1.3.Immune protection provided by IE-1 subunit vaccine Mice were immunized twice via i.p.or i.n.route with different amount of rIE-1 protein.After immunization,they were challenged with a lethal dose(3 × LD50)of highly virulent SG-MCMV.Vaccination could induce IE-1 specific humoral and cellular immune responses.The levels of both had a positive correlation with the vaccine dosage.Compared with the unimmunized control,the immunized mice had less body weight loss and spleen virus titer.The highest survival rate in intraperitoneally immunized mice was 87.5%,and that in intranasally immunized mice was only 25%,indicating that i.p.route was superior to i.n.route for vaccination of rIE-1 protein.Meanwhile,in order to enhance the immune effect,combinations of rIE-1 protein with MF59 and with CTB~* were used to immunize mice via i.p.and i.n.routes,respectively.Compared with the vaccine alone(via i.p.and i.n.routes),rIE-1 protein adjuvanted with MF59(via i.p.route)and rIE-1 protein adjuvanted with CTB~*(via i.n.route)resulted in significantly higher IgG antibody titers and IFN-γ secreting CD8+ T cell numbers,and also better protective abilities.The virus titers in spleens were significantly reduced but the residue virus could still be detectable.rIE-1 protein + MF59(via i.p.route)provided for mice 100% survival rate,but rIE-1 protein + CTB~*(via i.n.route)only provided less than 50% survival rate.The experiments demonstrated that: 1)rIE-1 had good immunogenicity;2)Addition of MF59 or CTB~* in the protein significantly enhanced the IE-1 specific humoral and cellular immune responses;3)Immunization with the protein via i.p.route provided better protection than via i.n.route.rIE-1 + MF59 was able to provide complete protection for mice.4.Immune protection provided by IE-1 DNA vaccine and subunit vaccine Mice were immunized once with IE-1 DNA(group D1),with IE-1 protein(group P1),primed with IE-1 DNA and subsequently boosted with the protein(group D1P1),both primed and boosted with the protein(group P2),respectively.The DNA was delivered through intramuscular injection followed by electroporation and the protein was delivered by i.p.or i.n.immunization.Mice were challenged with a lethal dose(3LD50)of SG-MCMV after immunization.Mice in D1 group had a relatively high number of spleen CD8+ T cells and 37.5% of them survived the lethal challenge.When rIE-1 protein was delivered by i.p.immunization,the survival rate and CD8+ T cell number among the groups were P1<P2< D1P1,and the spleen virus titer was P1>P2>D1P1.The CD8+ T cell number in D1P1 was significantly higher than those in P1 and P2,and the survival rate in D1P1 was 100% and in P2 was 75%,both significantly higher as compared with the unimmunized control(0%).When rIE-1 protein was delivered by i.n.immunization,the survival rates in all the tested groups had no significant difference with the control.Mice in D1P1 had the highest survival rate(50%).The CD8+ T cell number among the groups was P1<P2< D1P1,and the spleen virus titer was P1>P2>D1P1.The spleen virus titers between D1P1 and P1 had significant difference.The experiments demonstrated that,compared with immunization with IE-1 DNA or the protein alone,heterologous prime-boost immunization with IE-1 DNA and the protein was able to offer better immune protection for mice against the lethal virus challenge.5.Immune protection in mice provided by five DNA vaccines expressing different antigens Five DNA vaccines were constructed.Four encoding MCMV proteins gp34(m04),p65(M84),DNA helicase(M105),and immediate-early 1 protein pp89(IE-1),respectively,which were reported to induce CD8+ T cell responses,were compared with the one expressing gB(M55),the neutralizing antibody target antigen,for immune protection in BALB/c mice.Mice were immunized with these DNA vaccines,respectively,1 to 4 times via intramuscular injection followed by electroporation,and were subsequently infected with a lethal dose(3 × LD50)of highly virulent SG-MCMV.Mice immunized with more than one dose of M84 vaccine or two doses of m04 orIE-1 vaccine had a survival rate of 100%,and immunized with M55 or M105 DNA at four doses had only 62.5% survival rate.These DNA vaccines could effectively reduce the virus loads in salivary glands and spleens of mice,but couldn’t completely clear the residual virus.The experiments demonstrated that these DNA vaccines could effectively afford mice protection against infection with a highly virulent MCMV and that the protection offered by induced CD8+ T cell immunity might be superior to that by gB-specific antibodies.The above studies are valuable references for development and application of HCMV vaccines.