Induction of high level of specific antibody response to the neutralizing epitope ELDKWA on HIV-1 gp41 by peptide-vaccine☆

Peptides 21 (2000) 463– 468

Induction of high level of specific antibody response to the neutralizing epitope ELDKWA on HIV-1 gp41 by peptide-vaccine夞 Maofu Liaoa, Yun Lua, Yi Xiaoa, Manfred P. Dierichb, Ying-Hua Chena,* a

Laboratory of Immunology, Research Centre of Medical Science and the School of Life Science and Engineering, Tsinghua University, Beijing 100084, Peoples Republic of China b Institute of Hygiene, University of Innsbruck, Ludwig–Boltzmann-Institute for AIDS-Research, A-6010 Innsbruck, Austria Received 10 September 1999; accepted 15 December 1999

Abstract The monoclonal antibody 2F5 recognizing the neutralizing epitope ELDKWA on the C-domain could neutralize 90% of the investigated HIV-1 isolates. Low levels of ELDKWA-epitope-specific antibodies were observed in HIV-1-infected individuals. To induce high levels of antibodies to ELDKW-epitope, C-domain peptide (P2) was conjugated with a carrier peptide (KGGG)7-K (K/G). P2-K/G-conjugate induced high level of antibodies in mice by titer 1:25 600 to ELDKWA-epitope. P2-K/G-BSA-conjugate induced antibody response to ELDKWA-epitope (1:320 – 6400) in mice. The ELDKWA-epitope-specific antibodies of 19.8 and 34.6 ␮g/per milliliter serum were isolated from two rabbit antiserums (1:25 600). The levels of ELDKWA-epitope-specific antibodies induced in rabbits were greater than 1 ␮g/ml, a level considered to confer long-term protection. These results demonstrate the potential role of the C-domain peptide of gp41 to develop an effective ELDKWA-based epitope/peptide-vaccine against HIV-1. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Epitope-vaccine; Neutralizing epitope; HIV-1 gp41

1. Introduction An effective vaccine is urgently needed to stop AIDSepidemic. The recombinant gp120 (rgp120) of HIV-1 has been used to develop an antibody-based subunit vaccine against HIV-1 in several laboratories. Unfortunately, Connor and co-workers provided direct experimental evidences that rgp120 subunit vaccine did not protect individuals from HIV-1 infection [7]. Moore reviewed problems of gp120 subunit vaccine, and found that the failure of this vaccine results from inducing very weak neutralization activity against representative primary viral isolates, and indicated that a new strategy for developing an effective vaccine must be chosen to break through these problems [15,16]. To break through these problems, we suggest epitope/peptidevaccine as a new strategy against HIV-1 infection [5]. Recent studies revealed that gp41 plays a very important 夞 This work was supported by the 863- and 973-Programs, NSFC39880043, the Ministry of Education of China, and the Kingdream Ltd. * Corresponding author. Tel.: ⫹86-10-6277-2267; fax: ⫹86-10-62772267. E-mail address: [email protected] (Y.H. Chen).

role in HIV-1 entry into the target cells and should be a potential component for developing an effective vaccine to protect against HIV infection [1,12]. The C-domain of HIV-1 gp41 proved to interact with a potential receptor protein [2– 4], implicating in the process of HIV-entry. Interestingly, the monoclonal antibody (mAb) 2F5 recognizing an epitope (ELDKWA) on the C-domain could inhibit the binding of gp41 to lymphocytes and monocytes [4], and neutralize HIV lab strains and even recently separated African, Asia, American, and European strains from clades A, B, and E. Most of the investigated viruses were neutralized by 90% [17,21]. Besides, the strong immunogenicity of SIV transmembrane protein gp32 (especially both domains) was demonstrated, and antibodies against both regions (homologous with the N- and C-domains on gp41) of gp32 could protect macaques from SIV infection [13,18], indicating that both domains could induce protective potency. Recent studies have provided experimental evidence that a fusion-competent vaccine with broad neutralization of primary isolates of HIV is associated with these fusion intermediates, which suggests that the N- and C-domains of gp41 in the fusion intermediates could contribute to the broad neutralization activity [12].

0196-9781/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 0 ) 0 0 1 7 9 - 0

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Fig. 1. Induction of antibodies recognizing ELDKWA-epitope in mouse by immunization with P2-K/G epitope/peptide-vaccine. AS1: antisera induced by immunization with P2-K/G epitope/peptide-vaccine; NS: pre-immune mouse serum. P2: P2-peptide; E/2F5: C-(ELDKWAG)4-peptide. The ELDKWA-epitopespecific antibodies and P2-specific antibodies in AS1 or in control serum (NS) were identified in ELISA-assay. Results from five mice per group were expressed as the mean. Figure with bars and curves shows the data from one of three separate experiments. P2: Cys-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu-Ser-Gln-AsnGln-Gln-Glu-Lys-Asn-Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala; E/2F5: Cys-Glu-Leu-Asp-Lys-Trp-Ala-Gly-Glu-Leu-Asp-Lys-Trp-Ala-Gly-Glu-Leu-Asp-Lys-TrpAla-Gly-Glu-Leu-Asp-Lys-Trp-Ala-Gly; K/G: Lys-Gly-Gly-Gly-Lys-Gly-Gly-Gly-Lys-Gly-Gly-Gly-Lys-Gly-Gly-Gly-Lys-Gly-Gly-Gly-Lys-Gly-Gly-Gly-LysGly-Gly-Gly-Lys.

Based on the fact that at least 80% HIV-1-infected children did not develop antibodies to ELDKWA-epitope [9], we wanted to induce high levels of antibodies to ELDKWepitope in mice and rabbits. Here, we report that epitopevaccines as a new strategy against HIV-1 could induce high level of neutralization antibodies recognizing ELDKWAepitope in animal experiments.

2. Methods The ELDKWA-tetramer-peptide E/2F5 [EnvIIIB aa669 – 674: C-(ELDKWAG)4], C-domain peptide P2 (aa646 – 674: C-TSLIHSLIEESQNQQEKNEQELLELDKWA) of gp41 and carrier peptide K/G [(KGGG)7-K] were commercially synthesized in Genemed Synthesis Inc. (South San Francisco, CA, USA). Peptide P2 was chemically linked to the carrier peptide K/G by MBS-method [6], and then to the carrier protein BSA [bovine serum albumin (BSA); obtained from Sigma, St. Louis, MO, USA] by glutaradehyde-method [10]. After conjugation process, the precipitates were clearly observed. Each

group of mice (n ⫽ 5) and rabbit (n ⫽ 3) were immunized subcutaneously (s.c.) in the foot pad of mice with 10 ␮g peptide and of rabbit with 80 ␮g peptide (in the conjugate) in Freund’s adjuvant complete (CFA) (1:1 ratio) at a final volume of 50 ␮l. Boosters were given in incomplete Freund’s adjuvant (IFA) on Days 14, 28, and 42. Serums were separated. Preimmune serums were collected before immunization. Epitope-specific antibodies in serum or eluates were detected in the enzyme-linked immunosorbent assay (ELISA). Peroxidase-conjugated rabbit immunoglobulins (Ig) to mouse Ig (P0260) and goat anti-rabbit Ig (P0448) were obtained from Dako (Denmark). The peptides (5 ␮g/ml) were coated overnight on a microtiter plate at 4°C. Nonspecific binding was blocked by incubation with 1% BSA or 0.3% gelatin in PBS. After washing 3⫻ with PBS-Tween 20 (0.1% Tween 20), antisera, or normal serum (pre-immune serum) with different dilution were added and incubated for 1 h at room temperature. After washing, peroxidase-conjugated rabbit anti-mouse Ig or goat anti-rabbit Ig were added. After further washing, freshly prepared 2,2-azino-di-(3-ethylbenzthiazoline sulfonate)-peroxide solution was added and the optical density was measured.

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Fig. 2. Induction of antibodies recognizing ELDKWA-epitope in mouse by immunization with P2-K/G-BSA epitope/peptide-vaccine. AS2: antisera induced by immunization with P2-K/G-BSA epitope/peptide-vaccine; NS: pre-immune mouse serum. P2: P2-peptide; E/2F5: C-(ELDKWAG)4-peptide. The ELDKWA-epitope-specific antibodies and P2-specific antibodies in AS2 or in control serum (NS) were identified in ELISA-assay. Results from five mice per group were expressed as the mean. Figure with bars and curves shows the data from one of three separate experiments.

The epitope-specific antibodies were isolated from rabbit antiserum by fast performance liquid chromatography (FPLC) (Amersham Pharmacia Biotech, Sweden) using C-(ELDKWAG)4-sepharose-column and standard method from Pharmacia. The quantity of proteins was calculated by the program on FPLC. In control, proteins were isolated from normal serum (pooled pre-serum) using C-(ELDK WAG)4- or C-(GPGRAFY)4-sepharose-columns. The ability of the isolated ELDKWA epitope-specific antibodies to bind to the recombinant envelope protein was tested in Western blot. Soluble gp41 (sgp41), the ectodomain of gp41, was subjected to electrophoresis in a 9.5% SDS-polyacrylamide gel (SDS-PAGE) under reducing conditions and electroblotted onto nitrocellulose. Before probing, blots were blocked in PBS with 1% dried milk, 0.1% NaN3. The binding of the isolated ELDKWA epitope-specific antibodies (60 ␮g/ml) was detected with peroxidaseconjugated goat-anti-rabbit Ig (1:500 dilution).

3. Results Epitope-vaccine is principally based on the ‘principal neutralizing epitope’ (PND) of antigens, and belongs to an

especial type of synthetic peptide-vaccine. The monoclonal antibody 2F5 recognizing the neutralizing epitope ELDKWA on the C-domain proved to have significant potency to neutralize 90% of the investigated HIV-1 isolates [17,21]. Nevertheless, at least 80% HIV-1-infected children did not develop antibodies to ELDKWA-epitope [9]. We suggested that epitope/peptide-vaccine as a new strategy against HIV-1 could induce high level of neutralization antibodies [5]. To conform this hypothesis in animal experiments, we prepared two conjugates (P2-K/G and P2-K/G-BSA) and tested their potency to induce high level antibodies recognizing the neutralizing epitope ELDKWA in mice and rabbits. P2-K/G-conjugate after vaccination could induce high level of antibodies in mice by about 1:25 600 to ELDKWApeptide and 1:51 200 to P2 peptide (Fig. 1). The antibody response to ELDKWA-epitope (1:3200 – 6400) and P2 peptide (1:25 600) were induced in mice by P2-K/G-BSAconjugate (Fig. 2). Moreover, ELDKWA-epitope-specific antibodies (dilution by 1:25 600) in rabbit serum induced by P2-K/G-BSA-conjugate could be also observed, and strongly interacted with the P2-peptide (Fig. 3). These results from mice and rabbit experiments indicate that the synthetic peptide P2 coupled on the K/G-carrier peptide or

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Fig. 3. Induction of antibodies recognizing ELDKWA-epitope in rabbit by immunization with P2-K/G-BSA epitope/peptide-vaccine. AS3: rabbit antisera induced by immunization with P2-K/G-BSA epitope/peptide-vaccine; NS: pre-immune rabbit serum. P2: P2-peptide; E/2F5: C-(ELDKWAG)4-peptide. The ELDKWA-epitope-specific antibodies and P2-specific antibodies in AS3 or in control serum (NS) were identified in ELISA-assay. Results from three rabbits per group were expressed as the mean. Figure with bars and curves shows the data from one of three separate experiments.

Fig. 4. Identification of binding of the ELDKWA-epitope-specific antibodies in two eluates to C-(ELDKWAG)4-peptide, P2-peptide and control-peptide (K/G) in ELISA-assay. The ELDKWA-epitope-specific antibodies were isolated from rabbit antisera by FPLC using C-(ELDKWAG)4-sepharose-column. Ab1 (A): ELDKWA-epitope- specific antibodies isolated from rabbit 1 (1:50 dilution is equal to 0.7 ␮g/ml antibodies); Ab2 (B): ELDKWA-epitope-specific antibodies isolated from rabbit 2 (1:50 dilution is equal to 0.4 ␮g/ml antibodies). E/2F5: C-(ELDKWAG)4-peptide; P2: P2-Peptide; K/G: control peptide (carrier peptide). E/2F5, P2 and K/G (5 ␮g/ml) were coated on ELISA-plate. The epitope-specific antibodies in eluates were diluted in a series of dilutions. Results from two rabbits were expressed as the mean. Figure with bars shows the data from one of two separate experiments.

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Fig. 5. Binding of isolated ELDKWA-epitope-specific antibodies to soluble gp41 (sgp41) in Western blot. Sgp41 was analyzed on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE) under reducing condition (9.5%) and Western blots using the ELDKWA-epitope-specific antibodies isolated from rabbit serum (lane A) and rabbit serum preimmunization (lane B). Lane C: molecular weight markers: phosphorylase-b (97 kDa), BSA (66.2 kDa), ovalbumin (42.7 kDa), carbonic anhydrase (31 kDa), and lyspzyme (14.4 kDa).

K/G plus BSA carrier protein could induce high level of antibodies recognizing the neutralizing epitope ELDKWA. The ELDKWA-epitope-specific antibodies were isolated from two rabbit antiserums (1:25 600) using C-(ELDK WAG)4-sepharose-column, and tested in ELISA and by SDS-PAGE. The results of silver staining of eluates and control antibodies (IgM and IgG) indicated that proteins in eluates are immunoglobulins (data not shown). It was also indicated that 19.8 and 34.6 ␮g ELDKWA-epitope-specific antibodies per milliliter serum were observed and identified to recognize C-(ELDKWAG)4-peptide in ELISA-assay (Fig. 4), whereas 0.0021 and 0.0069 ␮g proteins per milliliter normal serum (pooled pre-serum) isolated using C-(ELDKWAG)4- or C-(GPGRAFY)4-sepharose-columns were observed. The levels of ELDKWA-epitope-specific antibodies induced in rabbits were more than 1 ␮g/ml, a level considered to confer longterm protection [19]. Western blots revealed that the ELDKWA-epitope-specific antibodies could recognize the envelope protein gp41 (Fig. 5). These results from mice and rabbit experiments demonstrate that the synthetic peptide P2 coupled with K/G-carrier peptide or plus BSA could induce high level of antibodies recognizing neutralizing epitope ELDKWA. Such high levels of ELDKWA-epitope-specific antibodies induced in body indicate an important role of the C-domain peptide of gp41 to develop an effective ELDKWA-based epitope/ peptide-vaccine against HIV-1.

4. Discussion Several groups demonstrated that HIV-1 particles in blood of patients could be cleaned principally by the neutralizing antibodies [11]. Recent clinical investigation indicates that the antibody reactivity to ELDKWA-epitope on the C-domain of gp41 is associated with disease progression in children perinatally infected with HIV-1, and at least 80%

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of children have not detectable antibody reactivity to this epitope [9], indicating that the ELDKWA determinant could be an important component in the formulation of a vaccine. Sequence analysis of primary isolates suggests that the major determinant of mAb 2F5 binding corresponds to the amino-acid sequence LDKW. Naturally occurring and in vitro selected neutralization-resistant viruses contained changes in the D and K positions of the ELDKWA motif, and the amino-acid changes from D to N, D to E, and K to N caused abrogation of 2F5-binding to the ELDKWAepitope [20]. The restricted antigenic variability of the ELDKWA-epitope enables ELDKWA-epitope to be developed as an effective epitope/peptide-vaccine. We suggest epitope/epitope-vaccine as a new strategy against HIV-1 [5]. Ideal epitope-vaccine, in our strategy, will use a combination of several principals neutralizing epitopes. The efficacy of 100% neutralization of heterologous primary isolates may be achieved by inducing several neutralizing antibodies in a body, if these neutralizing epitopes, or in part, are presented on these primary isolates. In fact, in vitro, triple combination of human mAbs (2F5, 2G12, and IgG1b12) can neutralize primary HIV-1 strains at concentrations around 1000-fold lower than a polyclonal antibody preparation still being considered for passive immunotherapy [14]. More importantly, the speed to synthesize peptides is quicker than the speed of viral mutation. Hence, epitope/ peptide-vaccine as a new strategy may be used to develop an effective vaccine to induce high level of neutralization antibodies against HIV-1 or other viruses.

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