The Study Of Discovery And Functional Of Heavy Chain Antibody In HIV-infected Patients

1.Introduction As an important effector molecule in the humoral immune response,antibody plays an important role in body’s immune defense.In order to complete the secretion of antibody,two heavy chain polypeptides and two light chain polypeptides must be paired to form a tetramer.This assembly method has become a necessary condition for antibody secretion.However,the discovery of heavy chain antibody broke this classic theory of antibody secretion: Heavy chain antibody can be secreted independently in the absence of light chains.Heavy chain antibody mainly comes from two major groups: patients with heavy chain disease and camels.Heavy chain diseases are divided into ɑ-HCD,γ-HCD,and μ-HCD based on the type of heavy chain.HCAb in heavy chain diseases has long been regarded as a derivative product.Until the discovery of HCAb in camel serum,the proportion of HCAb in total camel serum antibody is as high as 50-75%,which is enough to show that HCAb plays a role in the immune defense system of camelid animals.Heavy chain antibody from the two sources generally has structural deletions.Camel HCAb lacks the CH1 domain.The missing parts of HCAb in heavy chain diseases include CH1 and VH domain.In the process of antibody synthesis,after the heavy chain polypeptide enters the endoplasmic reticulum,CH1 domain and its molecular chaperone Bi P are tightly bound,thereby preventing the secretion of heavy chain dimers.The deletion of CH1 domain in heavy chain diseases and camel HCAb is believed to affect the secretion of heavy chain antibody.Studies have shown that a type of IgH gene cloned from plasmablasts of patients with systemic lupus erythematosus can be independently expressed and secreted in mammalian cells.Unlike HCAb in camels and heavy chain diseases,this type of heavy chain antibody has complete VH and CH1 domains.Human immunodeficiency virus has infected more than 78 million people worldwide,and more than 39 million infected people have died of AIDS and other related diseases.As a very harmful infectious d isease,no radical cure or preventive vaccine has been developed so far.In recent years,there have been more and more reports of autoimmune clinical symptoms in HIV-infected persons.Autoreactive antibody has also been detected in HIV-infected persons,indicating that the process of HIV infection is accompanied by autoimmune diseases.At the same time,typical neutralizing antibody also exhibits polyreactivity/autoreactivity.Whether there is a connection between polyreactivity and neutralization ability,whether polyreactive antibody can provide high-efficiency immune protection,and whether polyreactive antibody could be used as neutralizing antibody for vaccine induction,polyreactive antibody have become a research hotspot in the field of anti-HIV infection.In summary,HIV infection is accompanied by autoimmune diseases,thus leading to the appearance of autoreactive antibody in patients.During the development of B cells,IgH that does not rely on Ig L expression and secretion may bypass the negative screening process of Ig L gene editing,and is finally secreted in the form of heavy chain dimers.The purpose of our research is to explore whether there are HCAbs in HIV-infected persons,and whether they have biological functions,to explore the potential factors affecting the secretion and expression of such heavy chain antibody and the secretion mechanism,to explore whether there is a relationship between polyreactive antibody and neutralizing antibody and whether autoreactive HCAb has the neutralizing ability,and to explore the potential mechanism and application value of HCAb during HIV infection.2.Methods(1)Western blotting: To detect the expression and expression level of IgH gene cloned from HIV-infected patients,and to analyze the effects of germline genotype on heavy chain antibody secretion.(2)ELISA: ds DNA,ss DNA,LPS,and insulin were used to detect the polyreactivity of HCAb;gp140T and gp41 were used to detect the affinity to HIV antigen.DWEYS was used to detect the recognition of neural antigens.(3)Immunofluorescence Staining: Hep-2 cells were used as antigens to analyze the autoreactivity of heavy chain antibody;neural SH-SY5 Y cells were used as antigens to analyze the autoreactivity of HCAb to nerve cells.(4)CCK8 experiment and Flow cytometry: SH-SY5 Y cells were used to analyze the interaction between HCAb and NMDAR-DWEYS.(5)Molecular construction: Analyzing the effects of CDR3 domain polyreactivity and autoreactivity by knocking out the CDR3 fragment of the heavy ch ain gene;by linking the heavy chain variable region of the CD4 i neutralizing antibody to the expression vector to analyze the neutralizing antibody biological properties of HCAbs.(6)Application of Hi Trap Lambda Fab Select and Hi Trap Kappa select magnetic beads: Using the characteristics of the two magnetic beads that can specifically recognize the bind to the constant region of the antibody light chain,and separate heavy chain antibody from the serum of HIV patients.3.Results(1)IgH gene expression analysis: The heavy chain genes cloned from plasmablasts of chronic HIV-infected patients were separately expressed,and it was found that heavy chain antibody can be independently expressed and secreted in the absence of light chain.After statistics on the secretion rate of heavy chain genes in the two groups,it was found the secretion rate of the control group is 23.23%,the HIV group is 74.39%.(2)Genotype analysis: Among these IgH genes,the secretion of heavy chain antibody is mainly determined by variable region genes.Statistical analysis of the genotype and secretion rate revealed that the VH3-23 genotype appeared the most frequently in secreted heavy chain antibody.(3)Subunit composition and mass spectrometry results: Denaturing non-reducing electrophoresis results showed that the molecular weight of the heavy chain antibody protein is about 110 k Da;denaturing reduction electrophoresis showed that the heavy chain antibody dissociates into a 55 k Da heavy chain polypeptide,indicating that HCAb is composed of two identical heavy chain polypeptides.Denaturing reduction electrophoresis also showed that the heavy chain antibody polypeptide and the complete antibody heavy chain polypeptide had the same size,indicating that this type of HCAb has a complete structure.The results of mass spectrometry showed that the heavy chain antibody has CH1 domain.(4)ELISA: HIV group HCAbs have obvious affinity to the four antigens of ds DNA,ss DNA,LPS and insulin.ELISA experiments showed that 73.3% of HCAbs are polyreactive;HCAbs have low affinity to HIV antigen gp140 T,gp41 has a high affinity,and ELISA results showed that 73.3% of HCAbs in the HIV group were anti-gp140.ELISA results showed that 73.3% of HCAbs in the HIV group were anti-DWEYS antigens.In the HIV group,66.7% of the heavy chain antibody has higher affinity for the above antigens than conventional antibody.(5)Immunofluorescence staining: HCAb can recognize Hep-2 nuclear and cytoplasmic component antigens,and the cellular immune response is s trong.A total of 80% of HCAb in the HIV group showed autoantibody.Heavy chain antibody can recognize SH-SY5 Y cell membrane surface antigens.(6)CCK8 and Flow cytometry: HCAb and NMDAR-DWEYS can cross-react,and can induce SH-SY5 Y cell apoptosis.(7)CDR3 knock-out experiment: CDR3 knock-out led to a severe decrease or even disappearance of the affinity of HCAbs to antigen.(8)HIV CD4 i plasmid construction: HCAbs of CD4 i neutralizing antibody showed low polyreactivity/self-reactivity and affinity for gp140.(9)Discovery of heavy chain antibody in HIV-infected patients: HCAbs were isolated from the serum of HV-infected persons.The ELISA results showed that the HCAbs in the serum have polyreactivity/autoreactivity,and obvious affinity for the gp140 and gp41.The comparison found that the anti-gp140 properties of HCAbs in serum of HIV patients were higher than that of recombinant HCAbs.4.Conclusion(1)The IgH gene cloned from plasmablasts of chronic HIV-infected persons can be independently expressed in mammalian cells without the light chain and secrete heavy chain antibody with a complete structure.This kind of HCAb not only breaks the classic antibody secretion theory in cell biology,but also challenges the secretion of camel heavy chain antibody to the outside of the cell only when the CH1 domain is missing,indicating that it affects the secretion of heavy chain antibody.The structure is not limited to the CH1 area.This HCAb may be secreted in a special way.(2)The heavy chain antibody cloned and expressed from the plasmablasts of HIV-infected persons is polyreactive/self-reactive.Self-HCAbs with biologically active indicate that the HIV infection process is accompanied by an autoimmune response.(3)This experiment confirmed that there is full-length heavy chain antibody in the serum of HIV-infected persons,and it is polyreactive and its anti-gp140 ability is significantly higher than that of recombinant HCAbs.The discovery of full-length HCAbs in the natural state is a very important su pplement to heavy-chain antibody.It not only breaks the secretion theory of heavy-chain antibody in the presence of CH1 region,the affinity for antigens also shows that full-length HCAb participates in the immune response.Polyreactivity and obvious anti-gp140 provide a new research sample for studying the correlation between polyreactive antibody and neutralizing antibody,as well as a new research idea for HIV vaccine design.