Antibody humanization is critical to reduce immunogenicity and enhance efficacy in the preclinical phase of the development of therapeutic antibodies originated from animal models.Computational suggestions have long b...Antibody humanization is critical to reduce immunogenicity and enhance efficacy in the preclinical phase of the development of therapeutic antibodies originated from animal models.Computational suggestions have long been desired,but available tools focused on immunogenicity calculation of whole antibody sequences and sequence segments,missing the individual residue sites.This study introduces Site-specific Immunogenicity for Therapeutic Antibody(SITA),a novel computational framework that predicts B-cell immunogenicity score for not only the overall antibody,but also individual residues,based on a comprehensive set of amino acid descriptors characterizing physicochemical and spatial features for antibody structures.A transfer-learning-inspired framework was purposely adopted to overcome the scarcity of Antibody-Antibody structural complexes.On an independent testing dataset derived from 13 Antibody-Antibody structural complexes,SITA successfully predicted the epitope sites for Antibody-Antibody structures with a receiver operating characteristic(ROC)-area unver the ROC curve(AUC)of 0.85 and a precision-recall(PR)-AUC of 0.305 at the residue level.Furthermore,the SITA score can significantly distinguish immunogenicity levels of whole human antibodies,therapeutic antibodies and non-human-derived antibodies.More importantly,analysis of an additional 25 thera-peutic antibodies revealed that over 70%of them were detected with decreased immunogenicity after modification compared to their parent variants.Among these,nearly 66%antibodies successfully iden-tified actual modification sites from the top five sites with the highest SITA scores,suggesting the ability of SITA scores for guide the humanization of antibody.Overall,these findings highlight the potential of SITA in optimizing immunogenicity assessments during the process of therapeutic antibody design.展开更多
African swine fever(ASF),caused by African swine fever virus(ASFV),is a highly contagious swine disease that has spread globally.Effective control strategies are not yet available.In this study,we prepared K205R mRNA,...African swine fever(ASF),caused by African swine fever virus(ASFV),is a highly contagious swine disease that has spread globally.Effective control strategies are not yet available.In this study,we prepared K205R mRNA,which was then formulated using Lipid Nanoparticle(LNP).The resulting K205R mRNA-LNP showed a particle size of approximately 86.27 nm and an mRNA encapsulation efficiency of 96.24%.Efficient expression of the K205R protein was confirmed in both HEK293T and PK15 cells.We further evaluated the immunogenicity of K205R mRNA-LNP in mice and pigs.All immunized animals developed significantly higher levels of IgG antibodies against K205R compared to the control group in the first week after the second immunization,with antibody titers reaching up to 105.Challenge experiments showed that K205R mRNA delayed the time of death.Our results suggested the successful implementation of the mRNA platform in the preparation and application of ASFV mRNA.展开更多
As PEGylated liposomes have witnessed remarkable advancements in drug delivery,their immunogenicity has emerged as a notable challenge.In this study,we discovered that a simple pre-injection of folic acid(FA)effective...As PEGylated liposomes have witnessed remarkable advancements in drug delivery,their immunogenicity has emerged as a notable challenge.In this study,we discovered that a simple pre-injection of folic acid(FA)effectively mitigated the immunogenicity of PEGylated liposomes and enhanced their in vivo performance by tolerating splenic marginal zone B cells.FA specifically inhibited the internalization of PEGylated liposomes by splenic marginal zone B cells,thereby reducing splenic lymphocyte proliferation and specific IgM secretion.This modulation alleviated Ig M-mediated accelerated blood clearance and adverse accumulation of the PEGylated liposomes in the skin.These findings provide new insights into the immunomodulatory effects of FA and promising avenues to enhance the efficacy and safety of PEGylated liposomal nanomedicines.展开更多
Objective The present study aimed to evaluate the immunogenicity of BA.2 variant receptor binding domain(RBD)recombinant protein formulated with CpG 1826 plus alum dual adjuvant.Methods The BA.2 variant RBD(residues 3...Objective The present study aimed to evaluate the immunogenicity of BA.2 variant receptor binding domain(RBD)recombinant protein formulated with CpG 1826 plus alum dual adjuvant.Methods The BA.2 variant RBD(residues 308-548)fusing TT-P2 epitope was obtained from prokaryotic expression system,purification technology and dialysis renaturation,which was designated as Sot protein.The soluble Sot protein formulated with CpG 1826 plus alum dual adjuvant was designated as Sot/CA subunit vaccine and then the BALB/c mice were intramuscularly administrated with two doses of the Sot/CA subunit vaccine at 14-day interval(day 0 and 14).On day 28,the number of effector T lymphocytes secreting IFN-γand IL-4 in mice spleen were determined by enzyme-linked immunospot(ELISpot)assay.The serum IgG,IgG1 and IgG2a antibodies were examined by enzyme-linked immunosorbent assay(ELISA).In addition,the level of neutralizing antibodies(NAbs)induced by Sot/CA subunit vaccine was also evaluated by the microneutralization assay.Results The high-purity soluble Sot protein with antigenicity was successfully obtained by the prokaryotic expression,protein purification and dialysis renaturation.The Sot/CA subunit vaccine induced a high level of IgG antibodies and NAbs,which were of cross-neutralizing activity against SARS-CoV-2 BA.2 and XBB.1.5 variants.Meanwhile,Sot/CA subunit vaccine also induced a high level of effector T lymphocytes secreting IFN-γ(635.00±17.62)and IL-4(279.20±13.10),respectively.Combined with a decreased IgG1/IgG2a ratio in the serum,which indicating Sot/CA subunit vaccine induced a Th1-type predominant immune response.Conclusion The Sot protein formulated with CpG 1826 plus alum dual adjuvant showed that the excellent cellular and humoral immunogenicity,which provided a scientific basis for the development of BA.2 variant subunit vaccines and references for the adjuvant application of subunit vaccines.展开更多
[ Objective ] The aim of the study was to construct associated DNA vaccine of PRRS （Porcine reproductive and respiratory syndrome） and PCV-2 （Porcine circovirus type 2） disease and study its immunogenicity. [ Meth...[ Objective ] The aim of the study was to construct associated DNA vaccine of PRRS （Porcine reproductive and respiratory syndrome） and PCV-2 （Porcine circovirus type 2） disease and study its immunogenicity. [ Method] In_ this study, the ORF5 gene of PRRSV isolated in Liaoning was cloned into plRES-neo expression vector, and the neo gene of plRES-neo expression vector was substituted by the ORF2 gene of the PCV-2 Mongolia strain to construct the recombinant expression vector. The expression in BHK cells was detected through Western blot and IFA. Then the ELISA antibody level and the number of spleen T lymphocytes were detected after Balb/c mice were immunized with this DNA vaccine. E Result] The recombinant plasmid plRES-ORF2-ORF5 was constructed successfully and could express the target proteins in BHK cells, as indicated by Western blot and IFA. There was no significant difference in ELISA antibody between plRES-ORF2-ORF5 immunized group and inactived vaccine immunized groups, while the number of spleen T lymphocytes induced by DNA vaccine was higher than that induced by inactived vaccine. [ Conclusion] The recombinant plasmid plRES-ORF2-ORF5 should induce good humoral immune response and cellular immune response in mice, providing the conditions for better prevention and control of PRRS and PCV-2 disease.展开更多
AIM： To construct a prokaryotic expression vector carrying Campylobacterjejuni peblA gene and express it in Escherichia coli. Immunoreactivity and antigenicity of rPEB1 were evaluated. The ability of rPEB1 to induce ...AIM： To construct a prokaryotic expression vector carrying Campylobacterjejuni peblA gene and express it in Escherichia coli. Immunoreactivity and antigenicity of rPEB1 were evaluated. The ability of rPEB1 to induce antibody responses and protective efficacy was identified. METHODS： peblA gene was amplified by PCR, target gene and prokaryotic expression ptasmid pET28a （＋） was digested with BamHI and XhoI, respectively. DNA was ligated with T4 DNA ligase to construct recombinant plasmid pET28a（＋）-peblA. The rPEB1 was expressed in E. coli BL21 （DE3） and identified by SDS-PAGE. BALB/c mice were immunized with rPEBI. ELISA was used to detect the specific antibody titer and MTT method was used to measure the stimulation index of spleen lymphocyte transformation. RESULTS： The recombinant plasmid pET28a （＋）-peblA was correctly constructed. The expression output of PEB1 protein in pET28a （＋）-peblA system was approximately 33% of total proteins in E. coil The specific IgG antibody was detected in serum of BALB/c mice immunized with rPEB1 protein. Effective immunological protection with a lower sickness incidence and mortality was seen in the mice suffering from massive C. jejuni infection. CONCLUSION： rPEB1 protein is a valuable candidate for C. jejuni subunit vaccine.展开更多
To purify the protein encoding the small capsid protein （SCP） of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi＇s sarcoma associated-herpesvirus （KSHV） was expressed in a prokaryoti...To purify the protein encoding the small capsid protein （SCP） of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi＇s sarcoma associated-herpesvirus （KSHV） was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf65 was induced by isopropyl-13-D-thiogalactopyranoside （IPTG） and the fusion protein was purified by chromatography. The expressed protein and its purified product were identified by sodium dodecyl sulfate- polyacrylamide gel electrophoresis （SDS-PAGE） and showed that 9 kDa was the expected size of the purified orf65 protein. The antiserum was produced in rabbit which was immunized by purified orf65 protein. An ELISA assay was established to analyze the immunogenicity of the purified orf65 protein. The ELISA analysis demonstrated that orf65 protein has strong immune activity, and the immune activity of polyclonal antibody against orf65 was more than 4 fold higher than that in the serum of the non-immunized rabbit. These results demonstrate that purified orf65 protein has very strong immunogenicity and can be used in screening KSHV infection in the general population using ELISA.展开更多
Enterovirus （EV71） can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 （VP1）, plays a critical role in the pathogenicity of EVT1. High level...Enterovirus （EV71） can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 （VP1）, plays a critical role in the pathogenicity of EVT1. High level expression and secretion ofVP 1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP 1 DNA vaccines, including wt-VP 1, tPA-VP 1, VP l-d, VP 1-hFc and VP 1 - mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different （P〈0.05）. In this study, we further investigated the protein lev- els of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc （hFc） to VP1. VP 1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VPI-hFc protein for additional studies.展开更多
Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprise...Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.展开更多
Objective Preliminary assessment of rabies virus neutralizing activity,safety and immunogenicity of a recombinant human rabies antibody(NM57)compared with human rabies immunoglobulin(HRIG)in Chinese healthy adults.Met...Objective Preliminary assessment of rabies virus neutralizing activity,safety and immunogenicity of a recombinant human rabies antibody(NM57)compared with human rabies immunoglobulin(HRIG)in Chinese healthy adults.Methods Subjects were randomly(1:1:1)allocated to Groups A(20 IU/kg NM57),B(40 IU/kg NM57),or C(20 IU/kg HRIG).One injection was given on the day of enrollment.Blood samples were collected on days-7 to 0(pre-injection),3,7,14,28,and 42.Adverse events(AEs)and serious AEs(SAEs)were recorded over a period of 42 days after injection.Results All 60 subjects developed detectable rabies virus neutralizing antibodies(RVNAs)(>0.05 IU/mL)on days 3,7,14,28,and 42.The RVNA levels peaked on day 3 in all three groups,with a geometric mean concentration(GMC)of 0.2139 IU/mL in Group A,0.3660 IU/mL in Group B,and0.1994 IU/mL in Group C.At each follow-up point,the GMC in Group B was significantly higher than that in Groups A and C.The areas under the antibody concentration curve over 0-14 days and 0-42 days in Group B were significantly larger than those in Groups A and C.Fifteen AEs were reported.Except for one grade 2 myalgia in Group C,the other 14 were all grade 1.No SAEs were observed.Conclusion The rabies virus neutralizing activity of 40 IU/kg NM57 was superior to that of 20 IU/kg NM57 and 20 IU/kg HRIG,and the rabies virus neutralizing activity of 20 IU/kg NM57 and 20 IU/kg HRIG were similar.Safety was comparable between NM57 and HRIG.展开更多
AIM： To compare the response of standard hepatitis B virus （HBV） vaccination between patients with chronic hepatitis C virus （HCV） infection and healthy individuals. METHODS： This is a prospective case-control s...AIM： To compare the response of standard hepatitis B virus （HBV） vaccination between patients with chronic hepatitis C virus （HCV） infection and healthy individuals. METHODS： This is a prospective case-control study. A total of 38 patients with chronic HCV infection and 40 healthy controls were included. Vaccination was performed by injection of 20μg recombinant HBsAg into the deltoid muscle at mo 0,1 and 6. Anti-HBs concentration was determined 3 mo after the last dose and compared between the two groups. The response pattern was characterized as （1） high-response when the anti-HBs antibody titer was 〉 100 IU/L, （2） low-response when the titer was 10-100 IU/L. and （3） no-response when the titer was 〈 10 IU/L. RESULTS： In the patient group, there were 10/38 （26.3%） non-responders, 8/38 （21.1%） Iow-responders and 20/38 （52.6%） high-responders. The corresponding values in the control group were 2/40 （5.0%）, 7/40 （17.5%） and 31/40 （77.5%）, respectively. The response pattern was statistically different between the two groups. In multivariate analysis, smoking was a significant confounder, while HCV infection lost its significant correlation with lower antibody response. CONCLUSION： Patients with chronic HCV infection tend to respond weakly to HBV vaccination compared to healthy individuals, though this correlation is not independent according to multivariate analysis.展开更多
Bone is known to have a natural function to heal itself.However,if the bone damage is beyond a critical degree,intervention such as bone grafting may be imperative.In this work,the fabrication of a novel bone scaffold...Bone is known to have a natural function to heal itself.However,if the bone damage is beyond a critical degree,intervention such as bone grafting may be imperative.In this work,the fabrication of a novel bone scaffold composed of natural bone components and polycaprolactone(PCL)using 3D printing is put forward.α1,3-galactosyltransferase deficient pigs were used as the donor source of a xenograft.Decellularized porcine bone(DCB)with attenuated immunogenicity was used as the natural component of the scaffold with the aim to promote bone regeneration.The 3D printed DCB-PCL scaffolds combined essential advantages such as uniformity of the interconnected macropores and high porosity and enhanced compressive strength.The biological properties of the DCB-PCL scaffolds were evaluated by studying cell adhesion,viability,alkaline phosphatase activity and osteogenic gene expression of human bone marrow-derived mesenchymal stem cells.The in vitro results demonstrated that the DCB-PCL scaffolds exhibit an enhanced performance in promoting bone differentiation,which is correlated to the DCB content.Furthermore,critical-sized cranial rat defects were used to assess the effect of DCB-PCL scaffolds on bone regeneration in vivo.The results confirm that in comparison with PCL scaffolds,the DCB-PCL scaffolds can significantly improve new bone formation in cranial defects.Thus,the proposed 3D printed DCB-PCL scaffolds emerge as a promising regeneration alternative in the clinical treatment of large bone defects.展开更多
A synthetic hybrid 45-peptide gene of Plasmodium falciparum (Pf), which encoded two CSP repeated peptides NANP and three merozoite peptides SPf83. 1, SPf55. 1 and SPf35. 1, was cloned in an expression vector pWR450-1,...A synthetic hybrid 45-peptide gene of Plasmodium falciparum (Pf), which encoded two CSP repeated peptides NANP and three merozoite peptides SPf83. 1, SPf55. 1 and SPf35. 1, was cloned in an expression vector pWR450-1, then the recombinant plasmid pWRA was introduced into the attenuated Salmonella typhimurium SL3261. When used as a live vaccine and administered orally (po), intravenously (iv) or intraperitoneally (ip),the recombinant strain was able to live in vivo and elicit specific humoral and cellular immunity in BALB/c mice and rabbits. As oral immunization is safe and effective, it is thought that the live recombinant Salmonella tyPhimurium vaccine may bring the Pf oral live vaccine a step nearer.展开更多
Classical swine fever （CSF） and porcine reproduction and respiratory syndrome （PRRS） are both economically important, highly contagious diseases of swine worldwide. To develop an effective vaccine to control these...Classical swine fever （CSF） and porcine reproduction and respiratory syndrome （PRRS） are both economically important, highly contagious diseases of swine worldwide. To develop an effective vaccine to control these two diseases, we constructed a recombinant adenovirus rAdV-GP52AE2, using a replication-defective human adenovirus serotype 5 as a delivery vector, to co-express the GP5 protein of highly pathogenic porcine reproduction and respiratory syndrome virus （PRRSV） and the E2 protein of classical swine fever virus （CSFV）. Foot-and-mouth disease virus （FMDV） 2A peptide was used as a linker between the GP5 and E2 proteins to allow automatic self-cleavage of the polyprotein. The GP5 and E2 genes were expressed as demonstrated by immunofluorescence assay and Western blotting. Immunization of mice resulted in a CSFV-neutralizing antibody titer of 1：128 and a PRRSV-neutralizing antibody titer of 1：16. The lymphoproliferative responses were detected by Cell Counting Kit-8 assay and the stimulation index of CFSV-specific and PRRSV-specific lymphocytes in the rAdV-GP52AE2 group was significantly higher than that in the negative control group. The results show that rAdV-GP52AE2 can induce both effective humoral and cell-mediated immune responses in mice. The protective efficacy of the recombinant virus against CSF was evaluated in immunized rabbits, which were protected from fever induced by challenge with C-strain. Our study provides supporting evidence for the use of FMDV 2A to develop a bivalent genetically-engineered vaccine.展开更多
Embryonic stem cells （ESC） are suggested to be immune-privileged, but they carry the risk of uncontrolled expansion and malignancy. Upon differentiation they lose their tumor-forming capacity, but they become immuno...Embryonic stem cells （ESC） are suggested to be immune-privileged, but they carry the risk of uncontrolled expansion and malignancy. Upon differentiation they lose their tumor-forming capacity, but they become immunogenic by the expression of a normal set of MHC molecules. This immunogenicity might trigger rejection after application in regenerative therapies. In this study MHC expression of and immune responses to endothelial derivatives of rat embryonic stem cell-like cells （RESC） under inflammatory conditions were determined in comparison to primary rat aortic endothelial cells （ECs）. Cellular as well as humoral allo-recognition was analyzed in vitro. In addition, im- mune reactions in vivo were assessed by allo-antibody production and determination of interferon-y （IFNy）-secreting alio-reactive T cells. RESC derivatives expressed low but significant levels of MHC class I, and no MHC class II. In response to IFNy MHC class I expression was enhanced, while class II transactivator induction failed completely in these cells; MHC class II expression remained consistently absent. Functionally, the RESC derivatives showed a reduced allo-stimulatory capacity, protection against humoral allo-recognition in vitro and a slightly diminished sus- ceptibility to cytotoxic T cell lysis. Furthermore, in vivo experiments demonstrated that these ceils do not trigger host immune reactions, characterized by no allo-antibody production and no induction of allo-reactive memory T cells. Our results show that endothelial derivatives of RESC have a distinctive reduced immunogenic potency even under inflammatory conditions.展开更多
The FlaA gene from Vibrio harveyi marker, was cloned into the eukaryotic expression with a short nucleotide sequence encoding the Flag vector pcDNA3.1（＋） （designated as pcFlaA）. Ninety grouper （Epinephelus awoar...The FlaA gene from Vibrio harveyi marker, was cloned into the eukaryotic expression with a short nucleotide sequence encoding the Flag vector pcDNA3.1（＋） （designated as pcFlaA）. Ninety grouper （Epinephelus awoara） were separated into three equal size groups. An experimental group was immunized with pcFlaA, Control I group was immunized with the vector pcDNA3.1（＋）, and Control 1I group was immunized with PBS. The expression of pcFlaA mRNA and protein was examined using reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemistry. We also evaluated the immunogenicity and protective efficacy of pcFlaA against V. harveyi by measuring the lymphocyte proliferation response and serum levels of specific antibody and conducting a bacterial challenge test. We successfully transfected the fish muscle with pcFlaA. The pcFlaA mRNA and protein was expressed in the muscle cells for up to one month following injection. The proliferation response of lymphocytes in fish immunized with pcFlaA was significantly higher than in control group II. Furthermore, the immunized fish generated specific antibody. The vaccination also resulted in significantly higher survival during the bacterial challenge test.展开更多
Rabies, a zoonotic disease, causes > 55,000 human deaths globally and results in at least 500 million dollars in losses every year. The currently available rabies vaccines are mainly inactivated and attenuated vacc...Rabies, a zoonotic disease, causes > 55,000 human deaths globally and results in at least 500 million dollars in losses every year. The currently available rabies vaccines are mainly inactivated and attenuated vaccines, which have been linked with clinical diseases in animals. Thus, a rabies vaccine with high safety and efficacy is urgently needed. Peptide vaccines are known for their low cost, simple production procedures and high safety. Therefore, in this study, we examined the efficacy of multi-epitope-based vaccine candidates against rabies virus. The ability of various peptides to induce epitope-specific responses was examined, and the two peptides that possessed the highest antigenicity and conservation, i.e., AR16 and h PAB, were coated with adjuvant canineGp96 and used to prepare vaccines. The peptides were prepared as an emulsion of oil in water(O/W) to create three batches of bivalent vaccine products. The vaccine candidates possessed high safety. Virus neutralizing antibodies were detected on the day 14 after the first immunization in mice and beagles, reaching 5–6 IU/m L in mice and 7–9 IU/m L in beagles by day 28. The protective efficacy of the vaccine candidates was about 70%–80% in mice challenged by a virulent strain of rabies virus. Thus, a novel multi-epitope-based rabies vaccine with Gp96 as an adjuvant was developed and validated in mice and dogs. Our results suggest that synthetic peptides hold promise for the development of novel vaccines against rabies.展开更多
Highly attenuated modified vaccinia Ankara（MVA） is sensitive to repeat freeze-thaw cycle and easy to lose activity. In order to make the activity of MVA vaccine remain stable during its manufacturing, storage, and a...Highly attenuated modified vaccinia Ankara（MVA） is sensitive to repeat freeze-thaw cycle and easy to lose activity. In order to make the activity of MVA vaccine remain stable during its manufacturing, storage, and administration, the lyophilization as a good option could be resorted to; through screening, the right stabilizer composition and its production procedure were obtained. The final moisture content of freezing-dried recombinant MVA-HIV vaccine was lower than 3%. It can be reconstituted quickly and shows regular physical appearance and stable potency. In vivo functional experiment, mice were divided randomly into the liquid vaccination group, the lyophilized vaccination group, and the control group. Having been DNA vaccine priming, the mice were boosted with a dose of 10^7 pfu MVA- HIV vaccine, which produced indistinguishable antibody titer and cytotoxic T-lymphocyte（CTL） level compared with those of liquid vaccination group （ P 〉 0.05 ）. These results demonstrate that lyophilized MVA vaccine can induce high immunogenicity in mice.展开更多
Porcine circovirus type 2(PCV2)is the main causative agent of porcine circovirus-associated diseases,and it causes substantial economic losses in the swine industry each year.It is crucial to develop an efective vacci...Porcine circovirus type 2(PCV2)is the main causative agent of porcine circovirus-associated diseases,and it causes substantial economic losses in the swine industry each year.It is crucial to develop an efective vaccine against the circulating strain PCV2d,which is prone to substantial degrees of mutation.In this study,a truncated form of fagellin(tFlic:85-111 aa)was inserted into the C-terminal sequence of 2dCap,and Western blotting results showed that recombinant Cap-tFlic VLPs were successfully expressed.Transmission electron microscopy(TEM)and dynamic light scattering(DLS)data indicated that purifed recombinant Cap-tFlic fusion proteins existed in the form of polymers and that tFlic could not afect the formation and internalization of VLPs.Integrated Cap-tFlic VLPs induced the expression of antigen presentation-related factors(MHC-II and CD86)by bone marrow-derived dendritic cells(BM-DCs),and the expression of TLR5-related factors(TNF-α)was dramatically elevated.Mice intramuscularly immunized with Cap-tFlic VLPs exhibited signifcantly higher levels of Cap-specifc antibodies and neutralizing antibodies than mice immunized with wild-type Cap VLPs.The data obtained in the current study indicate that Cap-tFlic may be a candidate for a subunit vaccine against PCV2 in the future.展开更多
The BALB/c mice were immunized with Hsp70 DNA and Hsp65 DNA vaccines in human Mycobacterium tuberculosis. Eight weeks after immunization, the eyeballs were removed, blood and spleen taken, and intraperitoneal macropha...The BALB/c mice were immunized with Hsp70 DNA and Hsp65 DNA vaccines in human Mycobacterium tuberculosis. Eight weeks after immunization, the eyeballs were removed, blood and spleen taken, and intraperitoneal macrophages were harvested. The lymphocytic stimulating index (SI) was used to measure the cellular proliferating ability and NO release to measure the phagocytic activity of the macrophages. With ELISA kit, the levels of interleukin-2 (IL-2) and interferon-γ (IFN-γ) in serum and the splenic lymphocytic cultured supernatant were detected. The results showed that after the mice were immunized with 100 μg/mouse of Hsp70 DNA vaccine intramuscularly, the splenic lymphocytic proliferating ability in the mice was significantly increased as compared with that in the control group, vector group and Hsp65 DNA vaccine group (P<0.01); The contents of NO in the intraperitoneal macrophages of the mice were significantly lower than in the control group and Hsp65 DNA vaccine group (P<0.01); The levels of serum IL-2 in the mice were significantly higher than in the control group, but there was no statistical difference between Hsp65 DNA group and vector group (P>0.05); The contents of serum IFN-γ in the mice were significantly higher than in the control group, but significantly lower than in the Hsp65 DNA vaccine group (P<0.05). It was indicated that immunization with Hsp70 DNA vaccine could obviously enhance the immune response, but its intensity seemed inferior to Hsp65 DNA vaccine. The anti-infection mechanisms and clinical use in the future of the vaccines of Hsp70 DNA and Hsp65 DNA are worth further studying.展开更多
基金supported by funding from the National Key R&D Program of China(Grant Nos.:2023YFC3404000 and 2019YFA0905900)the National Natural Science Foundation of China(Grant Nos.:32370697 and 32070657)AI for the Science project of Fudan University,China(Project No.:XM06231724)。
文摘Antibody humanization is critical to reduce immunogenicity and enhance efficacy in the preclinical phase of the development of therapeutic antibodies originated from animal models.Computational suggestions have long been desired,but available tools focused on immunogenicity calculation of whole antibody sequences and sequence segments,missing the individual residue sites.This study introduces Site-specific Immunogenicity for Therapeutic Antibody(SITA),a novel computational framework that predicts B-cell immunogenicity score for not only the overall antibody,but also individual residues,based on a comprehensive set of amino acid descriptors characterizing physicochemical and spatial features for antibody structures.A transfer-learning-inspired framework was purposely adopted to overcome the scarcity of Antibody-Antibody structural complexes.On an independent testing dataset derived from 13 Antibody-Antibody structural complexes,SITA successfully predicted the epitope sites for Antibody-Antibody structures with a receiver operating characteristic(ROC)-area unver the ROC curve(AUC)of 0.85 and a precision-recall(PR)-AUC of 0.305 at the residue level.Furthermore,the SITA score can significantly distinguish immunogenicity levels of whole human antibodies,therapeutic antibodies and non-human-derived antibodies.More importantly,analysis of an additional 25 thera-peutic antibodies revealed that over 70%of them were detected with decreased immunogenicity after modification compared to their parent variants.Among these,nearly 66%antibodies successfully iden-tified actual modification sites from the top five sites with the highest SITA scores,suggesting the ability of SITA scores for guide the humanization of antibody.Overall,these findings highlight the potential of SITA in optimizing immunogenicity assessments during the process of therapeutic antibody design.
基金funded by the National Key Research and Development Program of China(2022YFD1800500,2021YFD1801401,2023YFD1802600)the Central Public-interest Scientific Institution Basal Research Fund,China(Y2022PT11)the Shanghai Sailing Program,China(23YF1457400).
文摘African swine fever(ASF),caused by African swine fever virus(ASFV),is a highly contagious swine disease that has spread globally.Effective control strategies are not yet available.In this study,we prepared K205R mRNA,which was then formulated using Lipid Nanoparticle(LNP).The resulting K205R mRNA-LNP showed a particle size of approximately 86.27 nm and an mRNA encapsulation efficiency of 96.24%.Efficient expression of the K205R protein was confirmed in both HEK293T and PK15 cells.We further evaluated the immunogenicity of K205R mRNA-LNP in mice and pigs.All immunized animals developed significantly higher levels of IgG antibodies against K205R compared to the control group in the first week after the second immunization,with antibody titers reaching up to 105.Challenge experiments showed that K205R mRNA delayed the time of death.Our results suggested the successful implementation of the mRNA platform in the preparation and application of ASFV mRNA.
基金supported by the National Natural Science Foundation of China(Nos.82373817 and 82003659)Shanghai Natural Science Foundation(No.23ZR1477500)Pudong Health Bureau of Shanghai(No.YC-2023-0401)。
文摘As PEGylated liposomes have witnessed remarkable advancements in drug delivery,their immunogenicity has emerged as a notable challenge.In this study,we discovered that a simple pre-injection of folic acid(FA)effectively mitigated the immunogenicity of PEGylated liposomes and enhanced their in vivo performance by tolerating splenic marginal zone B cells.FA specifically inhibited the internalization of PEGylated liposomes by splenic marginal zone B cells,thereby reducing splenic lymphocyte proliferation and specific IgM secretion.This modulation alleviated Ig M-mediated accelerated blood clearance and adverse accumulation of the PEGylated liposomes in the skin.These findings provide new insights into the immunomodulatory effects of FA and promising avenues to enhance the efficacy and safety of PEGylated liposomal nanomedicines.
基金funded by the National Key R&D Program of China(2023YFC2605302).
文摘Objective The present study aimed to evaluate the immunogenicity of BA.2 variant receptor binding domain(RBD)recombinant protein formulated with CpG 1826 plus alum dual adjuvant.Methods The BA.2 variant RBD(residues 308-548)fusing TT-P2 epitope was obtained from prokaryotic expression system,purification technology and dialysis renaturation,which was designated as Sot protein.The soluble Sot protein formulated with CpG 1826 plus alum dual adjuvant was designated as Sot/CA subunit vaccine and then the BALB/c mice were intramuscularly administrated with two doses of the Sot/CA subunit vaccine at 14-day interval(day 0 and 14).On day 28,the number of effector T lymphocytes secreting IFN-γand IL-4 in mice spleen were determined by enzyme-linked immunospot(ELISpot)assay.The serum IgG,IgG1 and IgG2a antibodies were examined by enzyme-linked immunosorbent assay(ELISA).In addition,the level of neutralizing antibodies(NAbs)induced by Sot/CA subunit vaccine was also evaluated by the microneutralization assay.Results The high-purity soluble Sot protein with antigenicity was successfully obtained by the prokaryotic expression,protein purification and dialysis renaturation.The Sot/CA subunit vaccine induced a high level of IgG antibodies and NAbs,which were of cross-neutralizing activity against SARS-CoV-2 BA.2 and XBB.1.5 variants.Meanwhile,Sot/CA subunit vaccine also induced a high level of effector T lymphocytes secreting IFN-γ(635.00±17.62)and IL-4(279.20±13.10),respectively.Combined with a decreased IgG1/IgG2a ratio in the serum,which indicating Sot/CA subunit vaccine induced a Th1-type predominant immune response.Conclusion The Sot protein formulated with CpG 1826 plus alum dual adjuvant showed that the excellent cellular and humoral immunogenicity,which provided a scientific basis for the development of BA.2 variant subunit vaccines and references for the adjuvant application of subunit vaccines.
文摘[ Objective ] The aim of the study was to construct associated DNA vaccine of PRRS （Porcine reproductive and respiratory syndrome） and PCV-2 （Porcine circovirus type 2） disease and study its immunogenicity. [ Method] In_ this study, the ORF5 gene of PRRSV isolated in Liaoning was cloned into plRES-neo expression vector, and the neo gene of plRES-neo expression vector was substituted by the ORF2 gene of the PCV-2 Mongolia strain to construct the recombinant expression vector. The expression in BHK cells was detected through Western blot and IFA. Then the ELISA antibody level and the number of spleen T lymphocytes were detected after Balb/c mice were immunized with this DNA vaccine. E Result] The recombinant plasmid plRES-ORF2-ORF5 was constructed successfully and could express the target proteins in BHK cells, as indicated by Western blot and IFA. There was no significant difference in ELISA antibody between plRES-ORF2-ORF5 immunized group and inactived vaccine immunized groups, while the number of spleen T lymphocytes induced by DNA vaccine was higher than that induced by inactived vaccine. [ Conclusion] The recombinant plasmid plRES-ORF2-ORF5 should induce good humoral immune response and cellular immune response in mice, providing the conditions for better prevention and control of PRRS and PCV-2 disease.
基金Grants from the Governor Foundation for Excellent Talents of Guizhou Province,No.200607
文摘AIM： To construct a prokaryotic expression vector carrying Campylobacterjejuni peblA gene and express it in Escherichia coli. Immunoreactivity and antigenicity of rPEB1 were evaluated. The ability of rPEB1 to induce antibody responses and protective efficacy was identified. METHODS： peblA gene was amplified by PCR, target gene and prokaryotic expression ptasmid pET28a （＋） was digested with BamHI and XhoI, respectively. DNA was ligated with T4 DNA ligase to construct recombinant plasmid pET28a（＋）-peblA. The rPEB1 was expressed in E. coli BL21 （DE3） and identified by SDS-PAGE. BALB/c mice were immunized with rPEBI. ELISA was used to detect the specific antibody titer and MTT method was used to measure the stimulation index of spleen lymphocyte transformation. RESULTS： The recombinant plasmid pET28a （＋）-peblA was correctly constructed. The expression output of PEB1 protein in pET28a （＋）-peblA system was approximately 33% of total proteins in E. coil The specific IgG antibody was detected in serum of BALB/c mice immunized with rPEB1 protein. Effective immunological protection with a lower sickness incidence and mortality was seen in the mice suffering from massive C. jejuni infection. CONCLUSION： rPEB1 protein is a valuable candidate for C. jejuni subunit vaccine.
基金Knowledge Innovation Program of the Chinese Academy of Sciences(0702121Y-J1)
文摘To purify the protein encoding the small capsid protein （SCP） of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi＇s sarcoma associated-herpesvirus （KSHV） was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf65 was induced by isopropyl-13-D-thiogalactopyranoside （IPTG） and the fusion protein was purified by chromatography. The expressed protein and its purified product were identified by sodium dodecyl sulfate- polyacrylamide gel electrophoresis （SDS-PAGE） and showed that 9 kDa was the expected size of the purified orf65 protein. The antiserum was produced in rabbit which was immunized by purified orf65 protein. An ELISA assay was established to analyze the immunogenicity of the purified orf65 protein. The ELISA analysis demonstrated that orf65 protein has strong immune activity, and the immune activity of polyclonal antibody against orf65 was more than 4 fold higher than that in the serum of the non-immunized rabbit. These results demonstrate that purified orf65 protein has very strong immunogenicity and can be used in screening KSHV infection in the general population using ELISA.
基金supported by the National Natural Science Foundation of China(Grant No.81000725 and 31470889)the Priority Academic Program of Basic Medical Science of Nanjing Medical University(Grant No.JX10131801060)
文摘Enterovirus （EV71） can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 （VP1）, plays a critical role in the pathogenicity of EVT1. High level expression and secretion ofVP 1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP 1 DNA vaccines, including wt-VP 1, tPA-VP 1, VP l-d, VP 1-hFc and VP 1 - mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different （P〈0.05）. In this study, we further investigated the protein lev- els of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc （hFc） to VP1. VP 1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VPI-hFc protein for additional studies.
基金National Basic Research Program ofChina (973 Program) (2009CB118701)National NaturalScientific Foundation of China (30671615, 30871940)+1 种基金Innovation Project of the Chinese Academy of Sciences(KSCX2-YW-N-021)Science and Technology Foundation of Zhejiang Province (2007C22052)
文摘Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.
基金supported by 2021 Beijing Key Specialty Program for Major Epidemic Prevention and Control。
文摘Objective Preliminary assessment of rabies virus neutralizing activity,safety and immunogenicity of a recombinant human rabies antibody(NM57)compared with human rabies immunoglobulin(HRIG)in Chinese healthy adults.Methods Subjects were randomly(1:1:1)allocated to Groups A(20 IU/kg NM57),B(40 IU/kg NM57),or C(20 IU/kg HRIG).One injection was given on the day of enrollment.Blood samples were collected on days-7 to 0(pre-injection),3,7,14,28,and 42.Adverse events(AEs)and serious AEs(SAEs)were recorded over a period of 42 days after injection.Results All 60 subjects developed detectable rabies virus neutralizing antibodies(RVNAs)(>0.05 IU/mL)on days 3,7,14,28,and 42.The RVNA levels peaked on day 3 in all three groups,with a geometric mean concentration(GMC)of 0.2139 IU/mL in Group A,0.3660 IU/mL in Group B,and0.1994 IU/mL in Group C.At each follow-up point,the GMC in Group B was significantly higher than that in Groups A and C.The areas under the antibody concentration curve over 0-14 days and 0-42 days in Group B were significantly larger than those in Groups A and C.Fifteen AEs were reported.Except for one grade 2 myalgia in Group C,the other 14 were all grade 1.No SAEs were observed.Conclusion The rabies virus neutralizing activity of 40 IU/kg NM57 was superior to that of 20 IU/kg NM57 and 20 IU/kg HRIG,and the rabies virus neutralizing activity of 20 IU/kg NM57 and 20 IU/kg HRIG were similar.Safety was comparable between NM57 and HRIG.
文摘AIM： To compare the response of standard hepatitis B virus （HBV） vaccination between patients with chronic hepatitis C virus （HCV） infection and healthy individuals. METHODS： This is a prospective case-control study. A total of 38 patients with chronic HCV infection and 40 healthy controls were included. Vaccination was performed by injection of 20μg recombinant HBsAg into the deltoid muscle at mo 0,1 and 6. Anti-HBs concentration was determined 3 mo after the last dose and compared between the two groups. The response pattern was characterized as （1） high-response when the anti-HBs antibody titer was 〉 100 IU/L, （2） low-response when the titer was 10-100 IU/L. and （3） no-response when the titer was 〈 10 IU/L. RESULTS： In the patient group, there were 10/38 （26.3%） non-responders, 8/38 （21.1%） Iow-responders and 20/38 （52.6%） high-responders. The corresponding values in the control group were 2/40 （5.0%）, 7/40 （17.5%） and 31/40 （77.5%）, respectively. The response pattern was statistically different between the two groups. In multivariate analysis, smoking was a significant confounder, while HCV infection lost its significant correlation with lower antibody response. CONCLUSION： Patients with chronic HCV infection tend to respond weakly to HBV vaccination compared to healthy individuals, though this correlation is not independent according to multivariate analysis.
基金the financial support from National Natural Science Foundation of China(81601626)Zhejiang Provincial Natural Science of Foundation of China(Y20C070010)+1 种基金start-up funding from Wenzhou Institute,University of Chinese Academy of Sciences(WIUCASQD2019002)Singapore MOE Tier 1 Grant RG46/18.
文摘Bone is known to have a natural function to heal itself.However,if the bone damage is beyond a critical degree,intervention such as bone grafting may be imperative.In this work,the fabrication of a novel bone scaffold composed of natural bone components and polycaprolactone(PCL)using 3D printing is put forward.α1,3-galactosyltransferase deficient pigs were used as the donor source of a xenograft.Decellularized porcine bone(DCB)with attenuated immunogenicity was used as the natural component of the scaffold with the aim to promote bone regeneration.The 3D printed DCB-PCL scaffolds combined essential advantages such as uniformity of the interconnected macropores and high porosity and enhanced compressive strength.The biological properties of the DCB-PCL scaffolds were evaluated by studying cell adhesion,viability,alkaline phosphatase activity and osteogenic gene expression of human bone marrow-derived mesenchymal stem cells.The in vitro results demonstrated that the DCB-PCL scaffolds exhibit an enhanced performance in promoting bone differentiation,which is correlated to the DCB content.Furthermore,critical-sized cranial rat defects were used to assess the effect of DCB-PCL scaffolds on bone regeneration in vivo.The results confirm that in comparison with PCL scaffolds,the DCB-PCL scaffolds can significantly improve new bone formation in cranial defects.Thus,the proposed 3D printed DCB-PCL scaffolds emerge as a promising regeneration alternative in the clinical treatment of large bone defects.
文摘A synthetic hybrid 45-peptide gene of Plasmodium falciparum (Pf), which encoded two CSP repeated peptides NANP and three merozoite peptides SPf83. 1, SPf55. 1 and SPf35. 1, was cloned in an expression vector pWR450-1, then the recombinant plasmid pWRA was introduced into the attenuated Salmonella typhimurium SL3261. When used as a live vaccine and administered orally (po), intravenously (iv) or intraperitoneally (ip),the recombinant strain was able to live in vivo and elicit specific humoral and cellular immunity in BALB/c mice and rabbits. As oral immunization is safe and effective, it is thought that the live recombinant Salmonella tyPhimurium vaccine may bring the Pf oral live vaccine a step nearer.
文摘Classical swine fever （CSF） and porcine reproduction and respiratory syndrome （PRRS） are both economically important, highly contagious diseases of swine worldwide. To develop an effective vaccine to control these two diseases, we constructed a recombinant adenovirus rAdV-GP52AE2, using a replication-defective human adenovirus serotype 5 as a delivery vector, to co-express the GP5 protein of highly pathogenic porcine reproduction and respiratory syndrome virus （PRRSV） and the E2 protein of classical swine fever virus （CSFV）. Foot-and-mouth disease virus （FMDV） 2A peptide was used as a linker between the GP5 and E2 proteins to allow automatic self-cleavage of the polyprotein. The GP5 and E2 genes were expressed as demonstrated by immunofluorescence assay and Western blotting. Immunization of mice resulted in a CSFV-neutralizing antibody titer of 1：128 and a PRRSV-neutralizing antibody titer of 1：16. The lymphoproliferative responses were detected by Cell Counting Kit-8 assay and the stimulation index of CFSV-specific and PRRSV-specific lymphocytes in the rAdV-GP52AE2 group was significantly higher than that in the negative control group. The results show that rAdV-GP52AE2 can induce both effective humoral and cell-mediated immune responses in mice. The protective efficacy of the recombinant virus against CSF was evaluated in immunized rabbits, which were protected from fever induced by challenge with C-strain. Our study provides supporting evidence for the use of FMDV 2A to develop a bivalent genetically-engineered vaccine.
文摘Embryonic stem cells （ESC） are suggested to be immune-privileged, but they carry the risk of uncontrolled expansion and malignancy. Upon differentiation they lose their tumor-forming capacity, but they become immunogenic by the expression of a normal set of MHC molecules. This immunogenicity might trigger rejection after application in regenerative therapies. In this study MHC expression of and immune responses to endothelial derivatives of rat embryonic stem cell-like cells （RESC） under inflammatory conditions were determined in comparison to primary rat aortic endothelial cells （ECs）. Cellular as well as humoral allo-recognition was analyzed in vitro. In addition, im- mune reactions in vivo were assessed by allo-antibody production and determination of interferon-y （IFNy）-secreting alio-reactive T cells. RESC derivatives expressed low but significant levels of MHC class I, and no MHC class II. In response to IFNy MHC class I expression was enhanced, while class II transactivator induction failed completely in these cells; MHC class II expression remained consistently absent. Functionally, the RESC derivatives showed a reduced allo-stimulatory capacity, protection against humoral allo-recognition in vitro and a slightly diminished sus- ceptibility to cytotoxic T cell lysis. Furthermore, in vivo experiments demonstrated that these ceils do not trigger host immune reactions, characterized by no allo-antibody production and no induction of allo-reactive memory T cells. Our results show that endothelial derivatives of RESC have a distinctive reduced immunogenic potency even under inflammatory conditions.
基金Supported by Fujian Science and Technology Innovation Foundation for Young Scientists (No.2006F3096)Scientific Research Foundation of Jimei University
文摘The FlaA gene from Vibrio harveyi marker, was cloned into the eukaryotic expression with a short nucleotide sequence encoding the Flag vector pcDNA3.1（＋） （designated as pcFlaA）. Ninety grouper （Epinephelus awoara） were separated into three equal size groups. An experimental group was immunized with pcFlaA, Control I group was immunized with the vector pcDNA3.1（＋）, and Control 1I group was immunized with PBS. The expression of pcFlaA mRNA and protein was examined using reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemistry. We also evaluated the immunogenicity and protective efficacy of pcFlaA against V. harveyi by measuring the lymphocyte proliferation response and serum levels of specific antibody and conducting a bacterial challenge test. We successfully transfected the fish muscle with pcFlaA. The pcFlaA mRNA and protein was expressed in the muscle cells for up to one month following injection. The proliferation response of lymphocytes in fish immunized with pcFlaA was significantly higher than in control group II. Furthermore, the immunized fish generated specific antibody. The vaccination also resulted in significantly higher survival during the bacterial challenge test.
基金supported by grants from the Special Fund for Agro-scientific Research in the Public Interest(201103032)the Fund of State Key Laboratory of Veterinary Biotechnology(SKLVBF201614)+3 种基金the National High Technology Research and Development Program of China(2015BAD11B02)Key Research Program of the Chinese Academy of Sciences(KSZD-EW-Z-005-001)the Special Fund for Chinese Academy of Sciences(CZBZX-1)WJL is the principal investigator of the NSFC Innovative Research Group(Grant No.81321063)
文摘Rabies, a zoonotic disease, causes > 55,000 human deaths globally and results in at least 500 million dollars in losses every year. The currently available rabies vaccines are mainly inactivated and attenuated vaccines, which have been linked with clinical diseases in animals. Thus, a rabies vaccine with high safety and efficacy is urgently needed. Peptide vaccines are known for their low cost, simple production procedures and high safety. Therefore, in this study, we examined the efficacy of multi-epitope-based vaccine candidates against rabies virus. The ability of various peptides to induce epitope-specific responses was examined, and the two peptides that possessed the highest antigenicity and conservation, i.e., AR16 and h PAB, were coated with adjuvant canineGp96 and used to prepare vaccines. The peptides were prepared as an emulsion of oil in water(O/W) to create three batches of bivalent vaccine products. The vaccine candidates possessed high safety. Virus neutralizing antibodies were detected on the day 14 after the first immunization in mice and beagles, reaching 5–6 IU/m L in mice and 7–9 IU/m L in beagles by day 28. The protective efficacy of the vaccine candidates was about 70%–80% in mice challenged by a virulent strain of rabies virus. Thus, a novel multi-epitope-based rabies vaccine with Gp96 as an adjuvant was developed and validated in mice and dogs. Our results suggest that synthetic peptides hold promise for the development of novel vaccines against rabies.
基金Supported by the National Natural Science Foundation of China(No30371317)
文摘Highly attenuated modified vaccinia Ankara（MVA） is sensitive to repeat freeze-thaw cycle and easy to lose activity. In order to make the activity of MVA vaccine remain stable during its manufacturing, storage, and administration, the lyophilization as a good option could be resorted to; through screening, the right stabilizer composition and its production procedure were obtained. The final moisture content of freezing-dried recombinant MVA-HIV vaccine was lower than 3%. It can be reconstituted quickly and shows regular physical appearance and stable potency. In vivo functional experiment, mice were divided randomly into the liquid vaccination group, the lyophilized vaccination group, and the control group. Having been DNA vaccine priming, the mice were boosted with a dose of 10^7 pfu MVA- HIV vaccine, which produced indistinguishable antibody titer and cytotoxic T-lymphocyte（CTL） level compared with those of liquid vaccination group （ P 〉 0.05 ）. These results demonstrate that lyophilized MVA vaccine can induce high immunogenicity in mice.
基金This work was supported by the Key Laboratory of Veterinary Biological Products and Chemicals,Ministry of Agriculture,China Animal Husbandry Industry Co.,Ltd.Project name:Novel Adjuvanted Chimeric Porcine Circovirus Type 2 CAP Virus-Like Particle Vaccine and project number:20201612(2020-2024).
文摘Porcine circovirus type 2(PCV2)is the main causative agent of porcine circovirus-associated diseases,and it causes substantial economic losses in the swine industry each year.It is crucial to develop an efective vaccine against the circulating strain PCV2d,which is prone to substantial degrees of mutation.In this study,a truncated form of fagellin(tFlic:85-111 aa)was inserted into the C-terminal sequence of 2dCap,and Western blotting results showed that recombinant Cap-tFlic VLPs were successfully expressed.Transmission electron microscopy(TEM)and dynamic light scattering(DLS)data indicated that purifed recombinant Cap-tFlic fusion proteins existed in the form of polymers and that tFlic could not afect the formation and internalization of VLPs.Integrated Cap-tFlic VLPs induced the expression of antigen presentation-related factors(MHC-II and CD86)by bone marrow-derived dendritic cells(BM-DCs),and the expression of TLR5-related factors(TNF-α)was dramatically elevated.Mice intramuscularly immunized with Cap-tFlic VLPs exhibited signifcantly higher levels of Cap-specifc antibodies and neutralizing antibodies than mice immunized with wild-type Cap VLPs.The data obtained in the current study indicate that Cap-tFlic may be a candidate for a subunit vaccine against PCV2 in the future.
基金This project was supported by a grant from National Natural Sciences Foundation of China(No. 39870 6 6 3)
文摘The BALB/c mice were immunized with Hsp70 DNA and Hsp65 DNA vaccines in human Mycobacterium tuberculosis. Eight weeks after immunization, the eyeballs were removed, blood and spleen taken, and intraperitoneal macrophages were harvested. The lymphocytic stimulating index (SI) was used to measure the cellular proliferating ability and NO release to measure the phagocytic activity of the macrophages. With ELISA kit, the levels of interleukin-2 (IL-2) and interferon-γ (IFN-γ) in serum and the splenic lymphocytic cultured supernatant were detected. The results showed that after the mice were immunized with 100 μg/mouse of Hsp70 DNA vaccine intramuscularly, the splenic lymphocytic proliferating ability in the mice was significantly increased as compared with that in the control group, vector group and Hsp65 DNA vaccine group (P<0.01); The contents of NO in the intraperitoneal macrophages of the mice were significantly lower than in the control group and Hsp65 DNA vaccine group (P<0.01); The levels of serum IL-2 in the mice were significantly higher than in the control group, but there was no statistical difference between Hsp65 DNA group and vector group (P>0.05); The contents of serum IFN-γ in the mice were significantly higher than in the control group, but significantly lower than in the Hsp65 DNA vaccine group (P<0.05). It was indicated that immunization with Hsp70 DNA vaccine could obviously enhance the immune response, but its intensity seemed inferior to Hsp65 DNA vaccine. The anti-infection mechanisms and clinical use in the future of the vaccines of Hsp70 DNA and Hsp65 DNA are worth further studying.
