The Immunogenicity Of Membrane Proximal External Region Immunogen From HIV-1 And Expression Of HIV-1 Broadly Neutralizing Antibodies Mediated By Recombinant Adeno-associated Virus 8

Human immunodeficiency virus type(HIV),which can causes acquired immune deficiency(AIDS),is a lentivirus that infects human immune system cells.Science HIV was discovered in 1983,the study to HIV has never stopped.But an effective vaccine against HIV has not been developed successfully.In this study,we aimed to elicit broadly neutralizing antibodies(Bn Abs).In the first part,the 10E8 Bn Ab that recognize the membrane proximal external region(MPER)of gp41 was used as the eliciting target.The MPER of gp41 is conserved in amino acid sequence,which can exposed during fusion of viruses and cells.MPER-derived peptide antigens,including the 10E8 epitope,are poorly immunogenic.Thus,Keyhole limpet hemocyanin(KLH),multiple antigen peptide(MAP),No V P particle were choosing as carriers for conjugating the 10E8 epitope,in order to induce MPER specific humoral immune response.The results showed that all immunogens could induce high levels of MPER-binding antibodies.Weak neutralizing activities could be detected in sera of guinea pigs.In recent years,towards the goal of elicting Bn Abs,different methods have been used,little success with only low neutralizing activity has been achieved in vaccines.Therefore,in the second part,a recombinant adeno-associated virus 8(r AAV8)vector was used to encode full-length antibodies against HIV-1 after gene transfer.10E8 m Ab targeting MPER or NIH45-46 m Ab recognizing CD4 bs was used as the target.The results showed that the Bn Abs could be expressed in the 293 T cell culture medium.A single intramuscular injection of r AAV8 led to more than 52 weeks expression of Bn Abs in Balb/c mice.Combined immunization with multiple Bn Abs has been attempted.The expressed antibodies in the supernatant of 293 T cells and in Balb/c mice showed neutralization against HIV-1 pseudoviruses.Combined immunization of r AAV8 expressing 10E8 and r AAV8 expressing NIH45-46 in Balb/c mice could increase these neutralization on strains of HIV-1 sensitive to 10E8 or NIH45-46 antibody compared with a single injection of r AAV8 expressing either antibody alone.Therefore,the combined immunization may be a potential vaccine approach against HIV-1.Part 1: The immunogenicity of membrane proximal external region immunogen from HIV-1Since the HIV-1 was discovered more than 30 years ago,measures toward an effective and safe HIV-1 vaccine have been attempted in a variety of strategies.However,little success was achieved.In recent years,a few broadly neutralizing antibodies(Bn Abs)have been isolated from HIV-1 elite controller.Select these epitopes that Bn Abs recognized to induce neutralizing antidodies is concerned by some researchers.10E8 Bn Ab has been found to be directed toward conserved epitope on the gp41 MPER,and lacks many of the characteristics previously thought to limit the usefulness of MPER-specific antibodies in vaccines,including autoreactivity.Therefore,design of different immunogens with 10E8 epitope to elicit 10E8-like Bn Abs were chosen in this study.MPER-derived peptide antigens,including the 10E8 epitope,are poorly immunogenic.Thus,KLH,MAP,No V P particle were choosing as an appropriate molecular carrier for conjugating the 10E8 epitope.The No V P monomer has three loops presented on the outermost surface of the No V P particle(PP),which are useful as epitope insertion points.In the current study,the No V P domain was designed as a single copy on each of the three loops or as three copies in loop 2.Therefore,the immunogen of 10E8-KLH,10E8-MAP4,10E8-loop123 PP and 10E8-3loop2 PP were designed.After the characterization of all immunogens,guinea pigs were immunized in order to obtain broadly nueralizing antibodies.The results showed that: MPER-specific antibody responses could be observed in all immuogen groups,which were enhanced following the fourth immunization,with comparatively high endpoint antibody titers(1 × 104–5).It is worth mention that the 10E8 epitope dispersed on three loops on the outermost surface of No V PPs(10E8-loop123 PP)elicited higher antibody titers than the equivalent number of epitopes presented on loop 2 only(10E8-3loop2 PP).However,in the subsequent neutralization experiment,no or very weak neutralization was elicited in the immune sera.The effective HIV-1 candidate vaccine was not obtained.In recent years,many of different vaccine candidates towards the Bn Abs of MPER have been tested in early phase human clinical trials with disappointed results.Therefore,a vaccine candidate for inducing HIV-1 Bn Abs remains to be developed.The disappointed results suggest that we need to change our mind to find a new way to obtain Bn Ab in vivo.Part 2: Expression of HIV-1 broadly neutralizing antibodies mediated by recombinant adeno-associated virus 8Gene therapy is used to carry foreign genes to achieve the purpose that cure some diseases.The gene therapy vectors contain viral vectors and non-viral vectors.However,non-viral vectors have low transfection efficiency and lack of targeting.Viral vectors have been widely used in gene therapy.The viral vectors that we always used include adenovirus,adeno-associated virus(AAV)and retrovirus.In order to achieve long-term expression of antibody,the recombinant AAV(r AAV)was used as the vector.AAV8 n Abs are more rare in human serum.Therefore,r AAV8 vector was used to transfer HIV-1 Bn Abs in this study.The CD4 binding site in HIV-1 could be recognized by CD4 T cells,while the MPER can be exposed before fusion of the virus membrane and T cell membrane.These are the major steps that allow HIV-1 to infect CD4 T cells.The NIH45-46 antibody which recognizes the CD4 binding site and 10E8 antibody targeting the MPER were mediated by r AAV8 vector.In this study,the HIV-1 antibody was driven from CMV or CAG promoter in order to obtain high antibody concentration.The 10E8 antibody driven by the CAG promoter was named CAG-10E8,and the NIH45-46 antibody under control of the CAG promoter was named CAG-NIH.The antibodies controlled by the CMV promoter were named in the same way as the CAG promoter,resulting in CMV-10E8 and CMV-NIH.The purified r AAV8 were evaluated by SDS-PAGE and TEM.The purified r AAV8 vectors expressing the HIV-1 antibodies were detected in 293 T cells.The heavy chain of antibody could be detected by Western blot and the nuetralization could be detected with HIV-1 pseudoviruses.Then administered by the intramuscular route to Balb/c mice for antibody expression with a single injection.The r AAV8 vector expressing the 10E8 or NIH45-46 antibody was immunized separately,and a novel combined immunization strategy of r AAV8 expressing 10E8 and r AAV8 expressing NIH45-46 was also designed.The antibody concentration of Balb/c mice sera were also evaluated by ELISA.The expressed antibodies of the CAG-10E8,CAG-NIH,CMV-10E8 and CMV-NIH groups could be maintained for 52 weeks.The novel combination of two r AAV8-mediated expressionof Bn Abs(10E8,NIH45-46)could also be detected in Balb/c mice sera.The expressed antibodies driven by the CAG promoter showed higher antibody concentration than those driven by the CMV promoter in vivo.The neutralizing activities could be detected in Balb/c mice.The neutralizing activities were found to be concentration-dependent,the combination of r AAV8 expressing 10E8 and r AAV8 expressing NIH45-46 showed stronger neutralization compared with a single injection of r AAV8 expressing either antibody alone in pseudovirus neutralization assays.In summary,we successfully expressed Bn Abs with r AAV8.After a single intramuscular injection of r AAV8 into Balb/c mice,the expression of the Bn Abs could be sustained for more than 52 weeks.This candidate HIV-1 vaccine may lay the foundation for further development.