GTMB 7 - Gene Therapy & Molecular Biology

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Arora and Seth: Antigenicity and immunogenicity of HIV envelope geneFigure 3. Autoradiograph showing dot blot analysis of celllysates from plaque picks infected Sf21 cells. 2 plaques labeledas P4 and P5 were found to be positive. Cells infected with wildtype baculovirus AcNPv served as the negative control. pBRUplasmid DNA served as the positive control.Figure 4. The photograph showing Immunofluorescencemicroscopy of the recombinant baculovirus infected Sf21 cells at48h-post infection. HIV-1 positive human polyclonal serumserved as the source of primary antibody.B. Expression of HIV-1 Envelopeglycoprotein by Recombinant BaculovirusExpre s sion of gp160 in Sf21 ce lls w a s exa mine d byindire ct immunofluores ce nce and w es te rn blot ana lysis ofinfec ted c e lls using HIV-1 pos itive huma n polyc lonal se ra .A 3+ fluore sc enc e wa s obs e rved at 48-hrs post infe c tion ona s ca le of 0 to 4+ that is from no fluore sc enc e to inte nsefluore sc enc e (Figur e 4). These results were supported bywestern blot analysis of the infected cells at 48hrs-postinfection. Gp160 and its cleavage products, Gp120 andGp41, could be detected after immunostaining. Since thetotal carbohydrate load added to the insect cell expressedglycoprotein is marginally less than that added duringsecretion from a mammalian cell, the baculovirusexpressed glycoprotein are correspondingly smaller (105kDa) than their mammalian counterparts (120 kDa) Nocorresponding protein bands were detected on from wildtype baculovirus (AcNPv) infected cells and uninfectedcells (Figure 5).Figure 5. Western blot analysis of recombinant baculovirusexpressed gp160. Lanes M: protein high range molecular weightmarker; 1: uninfected cell lysate; 2: cell lysate from AcNPvinfected cells; 3 & 4: cell lysate from recombinant baculovirusinfected cells.C. Lymphocyte Proliferation AssayIn vitro T cell proliferative activity of splenocytes fromanimals immunized with DNA vaccine pCIBRU-TREalone (group D3), boosted with P4 or vPE8/vPE16 (groupsD3P2, D3V2) or P4 and vPE8/vPE16 alone (groups D0P2,D0V2) was studied. (Table 1). Splenocytes from all theanimal groups showed positive proliferative response on invitro stimulation (Figure 6). Splenocytes from groupD0P2 mice demonstrated proliferation in response to P4cell lysate (SI-8.16), as well as to vPE8 and vPE16antigens (SI of 4 and 5.6). Splenocytes from DNA vaccineimmunized mice group D3 and D3P2 proliferated with SIof 8.8 on stimulation with vPE8 and with SI of 3.8 and 4.4respectively on stimulation with P4. Splenocytes frommice immunized with 2 doses of vaccinia expressedrecombinant Gp120/Gp160 with no DNA priming (GroupD0V2) showed better proliferation with vPE8, ascompared with vPE16 and P4. However, splenocytes frommice immunized with 3 doses of DNA followed by 2doses of vaccinia expressed recombinant Gp120/Gp160(Group D3V2) gave almost equal proliferation with P4,vPE8 and vPE16 respectively (Figure 6).Figure 6. In vitro T cell proliferative response to P4, vPE8 &vPE16 (recombinant baculovirus expressed gp160) ofsplenocytes from Balb/c mice immunized with pCIBRU-TRE (3doses at biweekly intervals) and boosted with 2 doses of eitherrecombinant baculovirus (P4) or recombinant vaccinia virus(vPE8 & vPE16). These groups of mice were marked as D3P2 orD3V2 respectively. Animals from groups D0P2 and D0V2 wereinjected only with recombinant baculovirus or recombinantvaccinia virus (no DNA priming).40
Gene Therapy and Molecular Biology Vol 7, page 41Table 1. Different groups of mice primed with pCIBRU-TRE DNA and boosted with baculovirus expressed (P4) orvaccinia expressed (vPE8 and vPE16) recombinant gp160.Group pCIBRU-TRE P4 vPE8 and vPE16D3D0P23 doses2 dosesD3P2 3 doses 2 dosesD0V22 dosesD3V2 3 doses 2 dosesIV. DiscussionThe main objective of this study was to prepare largeamounts of HIV-1 envelope protein, which may be used asa source of antigen for studying immune response againstHIV-1. HIV-1 gp160 with its signal sequence along withthe regulatory genes tat and rev was used to producerecombinant baculovirus (Malim et al, 1989; Ruben et al,1989 Rosen and Pavlakis; 1990, Roy et al, 1990). Thissystem has several advantages over other systemsincluding high level of protein production and posttranslationalmodification, which cannot be achieved inbacterial system (Luckow and Summers 1988, 1989). Weobserved poor expression of envelope proteins followinginfection of Sf21 cells as no protein was observed afterSDS-PAGE of the P4 infected Sf21 cell lysate followed bycoommassie blue staining. Several other studies haveindicated that env protein is refractory to efficientrecombinant expression (Lasky et al, 1986 Hu et al, 1987;Hu et al, 1987). Replacement of the signal sequence of theHIV-1 envelope protein with those of herpes simplex virusglycoprotein or human tPA results in efficient expression(Lasky et al, 1986; Berman et al, 1988). These studiestherefore suggest that the signal sequence of HIV-1envelope gene, which consists of 5 positively chargedamino acids, may be responsible for the poor expression.Li et al, (1994), showed that substitution of the gp120natural signal sequences with the signal sequences fromhoneybee mellitin or murine interleukin 3 promotes a highlevel of expression of a glycosylated form of gp120 andefficient secretion. These heterologous signal sequencescontain one (mellitin) or no (IL-3) positively chargedamino acids. These workers also demonstrated that onstepwise substitution of positively charged amino acidswith neutral amino acids resulted in enhanced expressionof HIV-1 gp120. Similarly, Golden et al, 1998, comparedthree different signal sequences [human tissueplasminogen activator (tPA), human placental alkalinephosphatase (pap), or baculovirus envelope glycoprotein(gp67)] and found that the tPA leader yielded the highestlevel of secreted protein, followed by the gp67 and papsequences.In this study, however, HIV-1 gp160 and its completecleavage products were observed on Western Blot analysisusing HIV-1 positive human polyclonal sera. Suggestingthereby that the envelope protein retained its antigenicityand may be used as a source of antigen for Western Blotanalysis. Immunogenicity as well as antigenicity of thisbaculovirus expressed envelope protein was alsodemonstrated by lymphocyte proliferation assays. Largescaleprotein purification is being pursued for furtherstudies.AcknowledgmentsThe Department of Biotechnology, Ministry ofScience and Technology, Government of India hasprovided financial support for this research. Ms AlkaArora received Research Fellowship from CSIR duringthis study.ReferencesArora A and Seth P (2001). Immuniz ation w ith H IV- 1 Subtype Bgp160-D NA Induces Spec if ic as we ll as c ross- rea ctive I mmuneRe sponses in Mice . Indian J Med Res 114, 1-9.Berman PW, Nunes WM and Haffar OK (1988) Expression ofmembrane-associated and secreted variants of gp160 ofhuman immunodeficiency virus type 1 in vitro and incontinuous cell lines. J Virol 62, 3135-42.Earl PL, Moss B and Doms RW (1991) Folding, interaction withGRP78-BiP, assembly, and transport of the humanimmunodeficiency virus type 1 envelope protein. J Virol 65,2047-55.Galli RA, Castriciano S, Fearon M, Major C, Choi KW, MahonyJ and Chernesky M (1996) Performance Characteristics ofRecombinant Enzyme Immunoassay To Detect Antibodies toHuman Immunodeficiency Virus Type 1 (HIV-1) and HIV-2and To Measure Early Antibody Responses inSeroconverting Patients. J Clin Microbiol 34, 999–1002.Golden A, Austen DA, van Schravendijk MR, Sullivan BJ,Kawasaki ES, Osburne MS (1998) Effect of promoters andsignal sequences on the production of secreted HIV-1 gp120protein in the baculovirus system. Protein Expr Purif 14, 8-12.Goudsmit J, Lange JMA, Paul DA, Dawson GJ (1987)Antigenemia and antibody titers to core and envelopeantigens in AIDS, AIDS-related complex, and subclinicalhuman immunodeficiency virus infection. J Infect Dis 155,558-60.Hill CM, Deng H, Unutmaz D, Kewalramani VN, Bastiani L,Gorny MK, Zolla-Pazner S, Littman DR (1997) Envelopeglycoproteins from human immunodeficiency virus types 1and 2 and simian immunodeficiency virus can use humanCCR5 as a co-receptor for viral entry and make direct CD4-dependent interactions with this chemokine receptor. J Virol71, 6296-304.Hu SI, Kosowski SG and Schaaf KF (1987) Expression ofenvelope glycoproteins of human immunodeficiency virus byan insect virus vector. J Virol 61, 3617-20.Johnson JE (1992) Detection of human immunodeficiency virustype 1 antibody by using commercially available whole-cellviral lysate, synthetic peptide, and recombinant proteinenzyme immunoassay systems. J Clin Microbiol 30,216–218.Kozarsky K, Penman M, Basiripour L, Haseltine W, Sodroski Jand Krieger M (1989) Glycosylation and processing of thehuman immunodeficiency virus type 1 envelope protein. JAcquir Immune Defic Syndr 2, 163-9.Kowalski M, Potz J, Basiripour L, Dorfman T, Goh WC,Terwilliger E, Dayton A, Rosen C, Haseltine W, Sodroski J41
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