| Hantavirus cause two acute viral infectious diseases, Hantavirus pulmonary syndrome(HPS) and hemorrhagic fever with renal syndrome (HFRS). HFRS covers a wide range inChina with high morbidity and mortality. Additionally, prevention of HFRS is difficultconsidering its wide sources of infection and various animal hosts. The pathogenesis ofHFRS is complicated and molecular details remains unclear. There are no effective drugsfor HFRS treatment and the inactivated vaccine still remains some problems. Therefore, itis quite necessary to perform more basic researches on Hantavirus.Hantavirus glycoprotein (GP) can mediate the fusion between viral envelope and host cellmembrane, and stimulate the body to produce neutralizing antibodies which have certain protective activity to infected animals or HFRS patients. Due to the high relation to viruspathogenicity and the activity of inducing anti-virus protective immunity, GP is one of themost important factors in studying Hantavirus. Both Gn and Gc proteins of Hantaviruscontain highly conserved N-glycosylation sites. Increasing studies demonstrate thatN-glycosylation is important not only for intracellular transport and proper folding of GP,but also for the structure and function definitions of epitopes. Additionally,N-glycosylation of GP is also critical for the formation of mature viral particles, thevirulence and the viral immune evasion. However, the glycosylation study of Hantavirusglycoprotein is mainly focused on the proper folding of GP itself and the influence on cellmembrane fusion, but the influence of glycosylation on the oligosaccharide chain structure,the antigenicity, the immunogenicity of GP and the GP-mediated viral infection remainsunclear.To solve the above problems, the N-glycosylation site on the envelope glycoprotein of theHantaan virus (HTNV)76-118strain was mutated by site-directed mutagenesis and theappropriate recombinant pseudovirus was constructed. The oligosaccharide chain structureof each recombinant pseudovirus glycoprotein was analyzed by lectin microarray. Theinfluence of each glycosylation site on infection was evaluated by comparing infectionability of each recombinant pseudovirus in Vero-E6cells. The humoral and cellularimmune responses were detected after the mice immunized with each recombinantpseudovirus and the protective effects against HTNV infection were analyzed, and thus theeffects of glycosylation on antigenicity and immunogenicity were estimated.【Methods and Results】1. Construction and comparison of the oligosaccharide chain structure of therecombinant pseudovirus expressing HTNV M gene or its mutants1.1The GP-encoding sequence of HTNV strain76-118was amplified by PCR andsite-directed mutagenesis was used to generate the five N-glycosylation site mutants(N134Q, N235Q, N347Q and N399Q on Gn, and N928Q on Gc). Then expression vectors carrying wild-type or mutant GP gene were constructed, and transfected into293T cells together with the packaging plasmids to generate recombinant pseudovirus,which contents wild-type GP (termed rLV-M) or GP with N-glycosylation site mutant(termed rLV-M1, rLV-M2, rLV-M3, rLV-M4and rLV-M5, respectively) with the GFPreporter gene.1.2Oligosaccharide chain structure of each recombinant pseudovirus glycoprotein wasanalyzed by lectin microarray, and lectin-binding profiles of the six recombinantviruses were as follows: rLV-M bound to5kinds of lectins (WFA, STL, BS-I, DSAand WGA); rLV-M1bound to4kinds of lectins (STL, BS-I, DSA and WGA); rLV-M2bound to6kinds of lectins (STL, BS-I, DSA, WGA, NPL and PTL-II); rLV-M3bound to6kinds of lectins (WFA, STL, BS-I, DSA, NPL and PTL-II); rLV-M4boundto3kinds of lectins (STL, BS-I and DSA); rLV-M5bound to8kinds of lectins (AAL,GSL-I, STL, BS-I, DSA, BPL, NPL and PTL-II).1.3According to the differences in lectin-binding specificity, the different oligosaccharidechains on glycoprotein of recombinant pseudovirus could be revealed as follows: themain types of oligosaccharides on rLV-M were GalNAcβ1,4GlcNAc, GlcNAc, α-Gal,α-GalNAc, Multivalent Sia,(GlcNAc)n, β1-4GlcNAc and LacNAc; the main types ofoligosaccharides on rLV-M1were GlcNAc, α-Gal, α-GalNAc, Multivalent Sia,(GlcNAc)n, β1-4GlcNAc and LacNAc; the main types of oligosaccharides on rLV-M2were Non-substitutedα-1,6Man, α-Gal, α-GalNAc, GlcNAc, Multivalent Sia,(GlcNAc)n, β1-4GlcNAc, LacNAc and Gal; the main types of oligosaccharides onrLV-M3were GalNAcβ1,4GlcNAc, Non-substitutedα-1,6Man, α-Gal, α-GalNAc,GlcNAc, Gal, β1-4GlcNAc and LacNAc; the main types of oligosaccharides onrLV-M4were GlcNAc, β1-4GlcNAc, LacNAc, α-Gal and α-GalNAc; the main typesof oligosaccharides on rLV-M5were GlcNAc, β1-4GlcNAc, LacNAc, α-Gal,α-GalNAc, fucose, Galβ1-3GalNAc, Gal, Non-substitutedα-1,6Man andGalNAcα-Ser/Thr(Tn). Comparing with rLV-M, the oligosaccharide structures Galand Non-substitutedα-1,6Man appeared on rLV-M2, LV-M3and rLV-M5; the oligosaccharide structures Fucose, GalNAcα-Ser/Thr(Tn) and Galβ1-3GalNAcappeared on rLV-M5. However, the oligosaccharide structure GalNAcβ-1,4GlcNAcwas not appeared on rLV-M1, rLV-M2, rLV-M4and rLV-M5; Multivalent Sia and(GlcNAc)n were not appeared on rLV-M3, rLV-M4and rLV-M5.1.4The STL, BS-I and DSA lectins showed positive binding to all the six recombinantpseudovirus. The fluorescence signal intensities for lectin-binding were compared andresults were as follows: Compared to rLV-M, rLV-M1showed significant increasedbinding to all the three lectins (P<0.05); rLV-M2, rLV-M3and rLV-M4showedsignificant increased binding to BS-I (P<0.05); rLV-M2, rLV-M3, rLV-M4andrLV-M5showed significant decreased binding to DSA (P<0.05). No significantdifferences were found between other pairs of groups. These results indicate thatrLV-M2, rLV-M3and rLV-M4contain high level of α-Gal and α-GalNAc than rLV-M.However, the levels of β1-4GlcNAc and LacNAc are lower in rLV-M2, rLV-M3,rLV-M4and rLV-M5than in rLV-M. Especially, rLV-M1contains higher levels ofLacNAc, α-Gal, α-GalNAc, β1-4GlcNAc and GlcNAc than rLV-M.2. Comparative analysis of the infectivity and immunological characteristics of therecombinant pseudovirus expressing HTNV M gene or its N-glycosylation sitemutants2.1Vero-E6cells were infected by recombinant pseudovirus with same titers respectively.All the cells infected by recombinant pseudovirus displayed green fluorescence whenvisualized by fluorescence microscopy. Then, the GFP expression in cells was furtheranalyzed by flow cytometry.57.2%of cells infected with rLV-M were GFP positivewhile66.4%of cells infected with rLV-M3were GFP positive, which was10%higher than infected with rLV-M. In the cells infected with rLV-M1and rLV-M2, thepercentage of GFP positive cells was45.9%and48.5%respectively, both were10%lower than infected with rLV-M. Also,38.5%of cells infected with rLV-M4wereGFP positive,20%lower than infected with rLV-M. The percentage of GFP positive cells was lowest in the cells infected with rLV-M5, which was28.1%,50%lowerthan infected with rLV-M. Considering the analysis of the oligosaccharide chainstructure shown in part1, this result suggested that change of oligosaccharide chainstructure may affect the virus attachment protein (VAP) structure or the properfolding of glycoprotein and further lead to the change of infectious ability. The morenumbers and more complex structures of oligosaccharide chains added, the lowerinfectivity showed. The existence of GalNAcβ-1,4GlcNAc oligosaccharide chainswas one of the important factors that influence infectivity.2.2C57BL/6mouse was intramuscular immunized with each of recombinant pseudovirusrespectively and control pseudovirus rLV-ZsGreen, saline and inactivated vaccine ofHFRS was used as control. Humoral immune response in mice was detected byELISA and microneutralization test. The specific antibody against to HTNV GP couldbe detected by ELSIA in serum when immunizing mice with rLV-M, rLV-M1,rLV-M2, rLV-M3, rLV-M4and rLV-M5separately and geometric mean titers (GMT)of the antibody was134.5,123.4,134.5,134.5,123.4and134.5, respectively, with nosignificant difference between groups (P>0.05). In the mice immunized withinactivated vaccine, the GMT of antibody against GP in serum was146.7. Again,there was no significant difference comparing to the experimental groups (P>0.05).Neutralizing antibody in serum could be detected by microneutralization test whenimmunizing mice with rLV-M, rLV-M1, rLV-M2, rLV-M3, rLV-M4and rLV-M5separately, and GMT of those was103.7,103.7,95.1,113.1,95.1and87.2. Nosignificant difference was found between groups (P>0.05) but they were allsignificant higher comparing to the inactivated vaccine immunization group (P<0.05),the GMT of which was16.8.2.3Secretion of INF-γ, IL-2, IL-10and IL-4by spleen cells of immunized mice wasmeasured by ELISPOT method. Secretion of INF-γ, IL-2, IL-10and IL-4were allsignificant higher comparing to the saline group (P<0.05), but there was nosignificant difference among recombinant pseudovirus immunization groups (P>0.05). The secretion of INF-γ, IL-2from recombinant pseudovirus immunization mice weresignificant lower than those from inactivated vaccine immunization group (P<0.05),while comparing to rLV-ZsGreen immunization mice, there was no significantdifference (P>0.05). The secretion of IL-4, IL-10from recombinant pseudovirusimmunization mice were significant higher than rLV-ZsGreen immunization group(P<0.05) while there was no significant difference between recombinant pseudovirusimmunization groups and inactivated vaccine immunization group (P>0.05). To sumup, all recombinant pseudovirus containing HTNV GP could promote spleen cellssecreting more IL-4and IL-10, while INF-γ and IL-2production didn’t changecomparing to LV-ZsGreen immunization group. This result suggested that as antigen,recombinant pseudovirus containing HTNV GP induced immune response of host,which was due mainly to humoral immune response. Moreover, there was nosignificant difference of this effect between mutant and wild-type recombinantpseudovirus.2.4HTNV76-118virus was used to infect the C57BL/6mice immunized withrecombinant pseudovirus. Then HTNV antigen expression in liver and spleen of micefrom each immunization group was analyzed by ELISA. Comparing to therLV-ZsGreen immunization group and saline group, all recombinant pseudovirus canprotect the mice from HTNV infection to a certain extent (P<0.05) and there was nosignificant difference among recombinant pseudovirus immunization groups (P>0.05),but their ability to protect host from viral infection was weaker than inactivatedvaccine.【Conclusion】In this study, six HTNV recombinant pseudovirus were constructed by site-directedmutagenesis, including a pseudovirus expressing the wild-type GP (rLV-M) andpseudovirus expressing mutant GPs (rLV-M1, rLV-M2, rLV-M3, rLV-M4and rLV-M5).Lectin-microarray assays demonstrated that mutation of each glycosylation sites on Gn or Gc caused changes of oligosaccharide chain structure of the recombinant pseudovirus.Further analysis of infectivity showed the change of oligosaccharide chain structure affectthe VAP structure or the proper folding of glycoprotein and further result in the change ofthe infectious ability of pseudovirus. The more numbers and more complex structures ofoligosaccharide chains added, the lower infectivity of the pseudovirus showed. Theexistence of GalNAcβ-1,4GlcNAc oligosaccharide chains was one of the important factorsthat influence infectivity of recombinant pseudovirus. This result suggested that restrictionor modification of oligosaccharide chain structure could influence the infectivity ofrecombinant pseudovirus. Therefore, it may provide a new target molecule for preventionand cure of HTNV. Results from animal experiments showed that all recombinantpseudovirus containing HTNV GP could induce HNTV specific immune response of host,which was due mainly to humoral immune response and the mice immunized with theserecombinant pseudovirus could produce high-titer neutralizing antibodies. So, therecombinant pseudovirus containing HTNV GP could protect mice against HTNVinfection to a certain extent. Mutation of single glycosylation site had no obviousinfluence on immunogenicity of GP. It may be because of the changed oligosaccharidechain structure was far from immunodominant epitope of antigen or mutation of singleglycosylation site had little influence on spatial structure of epitope. Furthermore,recombinant pseudovirus expressing HTNV GP caused the host produce high-titerneutralizing antibodies. This result provides a good thought and experimental foundationfor further optimizing designation of genetically modified HTNV vaccine. |
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