Tsinghua Science and Technology Tsinghua University, China ISSN: 1007-0212 Vol. 6, Num. 3, 2001, pp. 260-264

Tsinghua Science and Technology, Vol. 6, No. 3, August 2001 pp. 260-264

Three Candidate Peptide-Vaccines in Combination To Induce High Levels of Multiantibodies Against HIV-1*

WANG Ying , TIAN Haijun , QIN Li ,  ZHU Mei , CHEN Yinghua

Laboratory of Immunology, Research Center for Medical Science, Department of Biological  Sciences and Biotechnology, Tsinghua University, Beijing 100084, China

*Supported  by the National Science Foundation for Outstanding Young Scientists of China (No.30025038), the National Key Basic Research Specific Funds(No.G1999054107), and the National Natural Science Foundation of China (No.39880043)
** To  whom correspondence should be addressed, E-mail:  chenyh@mail.tsinghua.edu.cn

Received: 2000-11-30

Code Number: ts01078

Abstract:   

N-and C-domains of human immunodeficiency virus type 1 (HIV-1) gp41 are demonstrated to play an important role in HIV-entry and prevention. In addition, the V3 loop on gp120 was identified as the principal neutralizing determinant (PND). Based on the fact that a combination of several monoclonal antibodies to different neutralizing epitopes showed great protection against intravenous challenge and vaginal transmission of pathogenic HIV-1/Simian immunodeficiency virus (SIV) chimeric virus on macaques, three candidate peptide-vaccines were prepared and used in combination to induce high levels of multiantibodies against HIV-1. The three peptides contained important functional regions on HIV-1 gp160. The N-domain peptide (P1: aa550-579) and C-domain peptide (P2: aa633-662) of gp41 and V3 peptide (P3: aa301-328) of gp120 were conjugated with bovine serum albumin (BSA) using the glutaraldehyde method. After the vaccination course, each of the three candidate peptide-vaccines induced strong antibody response in rabbits. The three vaccines used in combination induced high levels of multiantibodies against the peptides of the N-and C-domains and the V3 loop, with the titer of antibodies up to 1 : 6400-1 : 25 600 in rabbit sera in comparison with the titer of 1 : 800-1 : 3200 induced by rgp41 or rgp160. Our results indicate that immunogenicities of the N-and C-domains and the V3 loop in these three candidate peptide-vaccines were clearly stronger than those induced by rgp41 or rgp160, and these peptide-vaccines used in combination synchronously induced high levels of multiantibodies against HIV-1, suggesting that used in combination they may provide a new vaccine-strategy to induce strong multi-antiviral activity.

Key  words:  human immunodeficiency virus type 1 (HIV-1); peptide-vaccine; combination; immunogenicity

Introduction   

It has been demonstrated that human immunodeficiency virus type 1 (HIV-1) gp41 is involved in HIV-1 entry into the cell. Recent crystal structure analysis of gp41 revealed that the binding of gp120 to CD4 and a co-receptor induces the conformational changes of gp41, resulting in the exposure of the N-and C-domains and creation of a fusion intermediate and then the fusion process[1, 2]. It is generally agreed that the V3 loop of gp120 plays a major role in the binding of gp120 with the chemokine receptor[3-5]. Therefore, small molecular inhibitors of V3 could block HIV entry[6]. Neutralizing antibodies to the V3 loop confer protection to challenge infections[7, 8]. In addition, two hydrophobic heptad repeats (N-and C-domains) of the gp41 ectodomain showed pivotal function during the membrane fusion. Peptides corresponding to the helical regions in the gp41 core structure (DP107 corresponding the N-domain and DP178 in the C-domain) are potent inhibitors of HIV-1 infection[9]. The inhibition is derived from disruption of the formation of the fusion intermediate consisting of the N-and C-domains on gp41[2]. It has been demonstrated that antibodies induced by the peptides (homologous with the N-and C-domains on gp41) of the SIV transmembrane protein gp32 could protect macaques from SIV infection[10,11]. In addition, a fusion-competent vaccine with broad neutralization of primary isolates of HIV is associated with the fusion intermediates, which suggests that the N-and C-domains of gp41 in the fusion intermediates could contribute to the broad neutralization activity[12]. Studies by us and others provided experimental evidence that gp41 binds a potential cellular receptor via the N-and C-domains[13-16] , which could provide further understanding of the protective mechanism of the N-and C-domains based vaccine. Lately, some experimental evidences have been provided that some synthetic peptides, when conjugated with the proper carriers, can mimic the native or recombinant proteins to induce humoral and/or cellular immunity[17,18]. With these findings, we prepared three candidate peptide-vaccines containing N-and C-domains and V3 domain peptides to use them in combination to induce high levels of multiantibodies against HIV-1.  

1 Materials and Methods  

1.1 HIV-1 proteins, peptides and antibodies

The N-and C-domain peptides, P1 (LSGIV-QQQNNLLRAIEAQQHLLQLTVWGIK, aa550-579) and P2 (WMEWDREINNYTSLIHSLIEES-QNQQEKNE, aa633-662), and the V3 loop peptide P3 (CTRPNNNTRKSIRIQRGPGRAF-YTIGKI, aa301-328) were commercially synthesized by Genemed Synthesis Inc. (USA). P1, P2, and P3 were synthesized according to the sequences of HIV-1_IIIB . Peroxidase-conjugated swine immunoglulins (Ig) to rabbit Ig was obtained from Dako (Denmark). The recombinant gp41 (rgp41) and recombinant gp160 of HIV-1_IIIB were acquired from Biotest (Germany) and Bende Inc. (Austria).

 Rabbit antisera against rgp41 or rgp160 were prepared in our laboratory.

1.2 Construction of immunogen and  immunization 

Peptides of P1, P2, and P3 were separately coupled to bovine serum albumin (BSA, obtained from Sigma, USA) using the glutaraldehyde method[19]. Equal masses of conjugates were then mixed. Each group (n=3) of New Zealand white rabbits were immunized subcutaneously with each of three candidate peptide vaccines (100 mg/injection in Freund's adjuvant) in combination. Boosters were given in incomplete Freund's adjuvant (IFA) at 2-week intervals. Antisera were collected seven days after the second booster. Preimmune sera were collected before immunization.

1.3 Enzyme-linked immunosorbent assay  (ELISA)

The peptides (5 mg/mL) were coated overnight on microtiter plates at  4 °C . Nonspecific binding was blocked by incubation with  0.3%  gelatin in phosphate-buffered saline (PBS) for 3 hours. After washing three times with PBS-Tween 20  (0.05%) , the rabbit antisera or preimmune sera with different dilutions were added and incubated for one hour at room temperature. After washing, peroxidase-conjugated swine anti-rabbit antibodies were added. After further washing, freshly prepared o-phenylenediamine-peroxide solution was added and the optical density was measured at 450 nm.

1.4 Western blot analysis

Peptide-specific antibodies were also detected using western blot analysis. Rgp41 and PND-peptide (5 mg) were subjected to electrophoresis in an 11.5% SDS-polyacrylamide gel (SDS-PAGE) under reducing conditions, and then electroblotted onto nitrocellulose. Before probing, blots were blocked with  0.1%  gelatine, 1% dried milk,  0.05%  Tween-20. Then the diluted antisera and preimmune sera were added. After washing with PBS, binding of specific antibodies of rabbit was detected with peroxidase-conjugated swine anti-rabbit antibodies (1 : 1000 dilution).

2 Results and Discussion  

Over the last few years, the N-and C-domains of gp41 have been demonstrated to contribute to the broad neutralization activity[12] , with PND located inside the V3 loop of gp120 demonstrated to be the major component for inducing protective immunity against HIV-1[20]. In this study, three candidate peptide-vaccines containing the N-and C-domain peptides and the PND-peptide were prepared and used alone or in combination to induce high levels of multiantibodies against HIV-1.

Three candidate peptide-vaccines (P1-BSA, P2-BSA and P3-BSA) used alone or in combination induced strong antibody response to the N-domain peptide P1 (antisera titer: 1 : 6400-1 : 12 800) (Fig.1), the C-domain peptide P2 (antisera titer: 1 : 25 600) (Fig.2) and the V3 loop peptide P3 (antisera titer: 1 : 6400) (Fig.3), which were clearly stronger than the antibody responses induced by rgp41 or rgp160 (antisera titer: 1 : 800-1 : 1600) (Figs.1, 2, 3). In addition, immune blotting revealed that the antisera induced by these three candidate peptide-vaccines used in combination could recognize the rgp41 and V3 loop peptide, while the pre-immune serum did not (Fig.4). The antibody response to rgp41 and rgp160 induced by the peptide-vaccines in combination reached antisera titer of 1 : 6400-1 : 12 800  and 1 : 6400 (Fig.5). The data demonstrates that the immunogenicities of the N-and C-domains and the V3 loop on rgp41 or rgp160 are weak and that the peptide-vaccines can increase the immunogenicities of these three functional regions in comparison with rgp41 or rgp160. Therefore, these three candidate peptide-vaccines used in combination can synchronously induce high levels of multiantibodies against HIV-1, without significant interference with each other. These CM(22 findings suggest that the peptide-vaccines in combination may be a new vaccine-strategy to induce strong multiantiviral activities for prevention or immune therapy against HIV-1 infection.