Study On The Long-term Stable Secretion Of HIV Specific Antibodies By Lactobacillus

Objective: Over the past 30 years,AIDS has spread throughout the world and has become one of the major infectious diseases that endanger human health.Despite the increased use of the Highly Active Anti-Retroviral Therapy(HAART)to control the global spread of the HIV infection,daily administration of HAART brought a certain metal and economic burden to the patients.Additionally,the potential drug resistance,daily adherence and severe adverse reactions of drugs also affect the application of antiviral drugs.More importantly,due to the existence of the viral reservoir,the long–term use of the best drug of HAART is still unable to cure the HIV infection.Therefore,to develop an effective prophylactic vaccine against HIV still will be the urgent need worldwide.In the past,although a number of HIV vaccine projects have been advanced to clinical trials,none were successful.In the process of long-term HIV vaccine research,the researchers isolated many HIV broadly neutralizing antibodies,and a number of passive immunization treatments based on these antibodies or derivatives on HIV prevention and treatment have certain effect.Although compared with antiviral drugs,antibodies can play a role of antiviral time longer and don't need daily administration,in order to maintain an effective antiviral concentration for weeks,antibody administration for many times is still necessary.Therefore,the long-term existence of sufficient effective antibodies in the body,especially in the infected area(mucosa),is urgently needed for the HIV passive immunotherapy.Lactobacillus is commensal bacteria in human gastrointestinal tract and female genital tract for food grade,and for its food safety and stability of colonization on the mucosal surface,it has been widely concerned and recognized that lactobacillus is the carrier of active vaccine and the expression vector of heterologous protein on the surface of mucous membrane.Using lactobacillus mucosa colonization feature,the engineered recombinant bacteria can make HIV-1 specific antibodies or the analogues along with the growth and reproduction of lactobacillus on the mucosal surface.The engineered recombinant lactobacillus is expected to be an important mean to further improve the HIV prevention effect of passive immunization based the neutralizing antibodies.In recent years,sexual transmission has become the main route of HIV infection.In China,the proportion of homosexual transmission of HIV increased significantly,and the male rectal mucosa became the first part of HIV attack,and also became a key step,where to block HIV infection effectively.In this study,in order to achieve effective in preventing HIV transmission,we combined the neutralizing antibodies or the analogues and the expression of the commensal lactobacillus plantarum WCFS1 strain on rectal mucosa to secrete the neutralizing antibodies to maintain the long-term,effective concentration on the HIV attack place,which can prevent HIV infection and reduce the administration frequency.This study will provide certain technical support for the passive immunization and prevention of HIV mucosal transmission.Methods: In the first part of this study,we construct and modified the expression system of lactobacillus plantarum WCFS1 to increase the secretion of the heterologous protein.On the basis of the high prevalence rate of HIV among MSM population,we select the commensal lactobacillus plantarum WCFS1 strain of rectal mucosa as the host stain,and the expression plasmids plp0373_Amy,which contains erythromycin resistant gene and the replication elements of lactobacillus and E.coli,as backbone plasmid.In order to the clone and reconstruction of plasmid using E.coli,first,we insert the ampicillin resistant gene in the backbone plasmid plp0373_Amy to construct the new plasmid(plp0373_Amy(Em/Amp))with resistant markers of both erythromycin and ampicillin.Further,in order to improve the expression of heterologous protein based on lactobacillus plasmid,we construct the new plasmid pldhl0373_Amy(Em/Amp)with homologous signal peptide and homologous promoter,and predicted the splice site of signal peptide using the network tool Signal P.For the HIV neutralizing antibodies,we choose two antibodies as parental antibodies,which bound to HIV gp120 v3 glycan and bound to CD4 molecular domain 2.Moreover,we also construct a bispecific antibody using two parental antibodies.Then we replace Amy A gene of the plasmid pldhl0373_Amy(Em/Amp)and plasmid plp0373_Amy(Em/Amp)with the single-chain variable fragments of the above three antibodies.In the second part of this study,we identify the growing activities of the recombinant stains and the phenotype and anti-viral ability of the supernatant of recombinant stains.Using ELISA and Western blot methods,we test the existence and phenotype of single-chain variable fragments in the supernatant.We test the neutralizing abilities and breadth using pseudovirus system.We test the OD600 value and the CFUs/ml of the bacteria solutions after 24 hours' culture and measure to compare the growing activities between wild WCFS1 strain and recombinant strains.In the third part of this study,we test the colonization ability of recombinant strains on rectal mucosal epithelial cells and the mucosal damage after the colonization of recombinant strains on rectal mucosal epithelial cells.In the normal rectum mucosal FHC cell model,the bacteria colonize the epithelial cells.In the first 24-hour interval,the cells are lysed for assessment of: 1)apoptosis by caspase3/7 assay;2)epithelia-associated colony forming unites(CFU)in MRS agar,and supernatants are collected for measurement of cytokine IL-1a,IL-1RA,IL-6,IL-8 and TNF-a.Results:1.We reconstructed the lactobacillus plantarum WCFS1 expression plasmid(pldhl0373_Amy A)with homologous signal peptide and homologous promoter,and acquired the recombinant WCFS1 strains that can secrete the single-chain variable fragments of HIV antibodies.2.All single-chain variable fragments of HIV antibodies were expressed by the recombinant strains and bound to gp120 or CD4.Lactobacillus-produced single-chain variable fragments inhibited HIV-1 pseudoviruses from the global panel in a neutralizing assay.3.The wild lactobacillus plantarum WCFS1 and the engineered recombinant WCFS1 strains expressing single-chain variable fragments showed the same stable colonization pattern.The wild lactobacillus plantarum WCFS1 and the engineered recombinant WCFS1 strains expressing single-chain variable fragments could induce the apoptosis,but the levels of the apoptosis were lower than positive control.The level of protective anti-inflammatory mediator(IL-1RA)remained constant for wild type and recombinant stains.The levels of the proinflammatory mediators(IL-1a,IL-6,IL-8 and TNF-a)expressed by recombinant strains were similar to wild type strain.Conclusions:1.The engineered recombinant Lactobacillus plantarum WCFS1 can express the single-chain variable fragments of HIV antibodies with biological activities in the supernatant.2.The engineered recombinant Lactobacillus plantarum WCFS1 can colonize the rectal mucosal epithelial cells and not induce the strain-specific apoptosis and significant immune-inflammatory.3.The engineered recombinant Lactobacillus plantarum WCFS1 in this study,as live carrier,can colonize on the mucosa to maintain the long-term and effective concentration of anti-viral proteins.4.This study provided a new model for the prevention of homosexual transmission of HIV by proposed recombinant lactobacillus.