Fine characterization of a V3-region neutralizing epitope in human immunodeficiency virus type 2
Introduction
Although it is debated which relative role the humoral immune response might play in protective immunity against human immunodeficiency virus (HIV), it is conceivable that the induction of broadly neutralizing antibodies would be a beneficial property of a future HIV vaccine. Anti-HIV antibodies generally appear in newly HIV infected individuals after 1–4 weeks (Gaines et al., 1987, Tindall et al., 1988; Lindbäck et al., unpublished results), although some individuals seroconvert after several months. HIV-1 isolate specific neutralizing antibodies appear rapidly, but are soon rendered ineffective due to emergence of virus variants resistant to neutralization by autologous, but not heterologous, sera (Albert et al., 1990, Tremblay and Wainberg, 1990, Von Gegerfelt et al., 1991). Furthermore, sera from HIV-infected individuals often show a strong neutralizing activity against common laboratory strains, while they fail to exhibit the same high titers of neutralizing antibodies against primary virus isolates. Viral factors suggested to be involved in the relative neutralization resistance of primary HIV isolates include a less accessible CD4 binding site on the primary virus surface glycoprotein as compared to the laboratory adapted (Moore et al., 1992) and a higher envelope spike density (Moore et al., 1993).
We have earlier shown that serum from HIV-2 infected patients, in contrast to HIV-1 infected individuals, retain the capability to neutralize autologous virus throughout the course of the infection (Björling et al., 1993), indicating a less efficient neutralization escape ability of HIV-2 as compared to HIV-1. There have also been reports of antibodies either neutralizing or enhancing HIV infection, depending on the viral strain used (reviewed by Levy, 1993).
Efforts have been made to identify which immunogenic sites are capable of eliciting broadly reactive neutralizing antibodies against HIV. It has been found in several studies that neutralizing antibodies in the early HIV-1 infection are primarily directed to the third variable region (V3) of the surface glycoprotein gp120 and the CD4 binding region (reviewed by Fenyö et al., 1996). The immunodominance of the V3 region is a major obstacle for an efficient humoral immune response, since its high sequence variability gives rise to variant viruses at such a high rate that the immune system is finally unable to recognize and produce neutralizing antibodies against new antigenic variants (reviewed by Coffin, 1986, Nara et al., 1990). However, anti-V3 murine monoclonal antibodies (Mabs) with virus neutralizing capacity have been obtained (Matsushita et al., 1988, Gorny et al., 1991, Ohno et al., 1991), and protection from infection has been achieved in chimpanzees by passive immunization with Mabs directed against the V3 region of HIV-1 (Emini et al., 1990). Primary isolates are generally less sensitive to neutralization by anti-V3 Mabs, as well as antibodies directed to other regions (Conley et al., 1994, Matthews, 1994, Moore et al., 1995). Several groups have reported that the GPGR motif located in the central part of the V3 loop of HIV-1 constitutes a relatively stable part of a principal neutralizing determinant (PND) (Javaherian et al., 1989, Gorny et al., 1993, Langedijk et al., 1992).
For HIV-2, the targets for neutralizing antibodies have only been partially identified, and characterization of the potential neutralizing activity of antibodies reacting with HIV-2 gp125 has produced conflicting results. There are reports that describe a failure to induce neutralizing antibodies to the HIV-2 V3 region by peptide immunization (Robert-Guroff et al., 1992, Babas et al., 1994). Using synthetic peptides corresponding to the V3-loop of HIV-2SBL6669-ISY, we have previously identified this region as an important target for neutralization of HIV-2 by raising neutralizing guinea pig anti-sera, by blocking neutralization activity of human HIV-2 sera and by developing neutralizing murine Mabs. Fine mapping has revealed two distinct antigenic sites with conserved motifs within the V3-loop; FHSQ (positions 315–318) and WCR (positions 329–331) (Björling et al., 1991, Björling et al., 1994). These two regions may interact as one discontinuous antigenic site. Mabs directed against the HIV-2 V3 region have also been obtained by other groups, both with (Matsushita et al., 1995, McKnight et al., 1996) and without (Traincard et al., 1994) neutralizing capacity.
The aim of this study was to further characterize the immunogenic sites in the V3-loop of HIV-2 important for eliciting neutralizing antibodies and study in more detail the importance of different V3 peptide configurations for their ability to elicit anti-HIV-2 neutralizing antibodies in a guinea pig model. We synthesized a panel of peptides of different lengths and with small variations in sequence, representing the central and C-terminal part of the HIV-2 V3 region, and tested them for recognition by a panel of human anti-HIV-2 sera. Guinea pigs were immunized with the peptides, and the reactivity of the resulting hyperimmune sera were tested against gp125 in Western blot analysis and analyzed for their neutralizing capacity against HIV-2SBL6669. We also evaluated the conformational changes induced by alanine substitutions in a peptide covering the FHSQ and WCR motifs by creating three dimensional models of the original and the mutated peptide, using the Monte Carlo simulation method.
Section snippets
Peptides
Forty-four peptides, 10–37 amino acids (aa) long, corresponding to the previously shown neutralizing V3 region in the envelope glycoprotein of HIV-2, were synthesized according to the solid-phase multiple peptide method (Table 1, Table 2) (Houghten, 1985), using t-Boc chemistry, in which the amino group is protected by an acid labile tert-butyloxycarbonyl group, which is removed before each coupling by treatment with TFA. The peptide amino acid sequences were derived from HIV-2SBL6669-ISY (
Results
To analyze the optimal design of a peptide for induction of HIV-2 neutralizing anti-V3-antibodies in guinea pigs, we synthesized 44 peptides, ranging from 10 to 37 aa of length. The peptides were chosen to cover the region of interest, i.e. the central and C-terminal part of the HIV-2SBL6669-ISY V3 region. Peptides ranging from 10 to 27 residues corresponding to this region were used in an attempt to optimize peptide sequence and length for induction of HIV-2 neutralizing antibodies (Table 1).
Discussion
In the present work we have studied the ability of peptides corresponding to the V3 region of HIV-2SBL6669-ISY, of different lengths and with various mutations, to elicit HIV-2 neutralizing antibodies in guinea pigs. We noted that subtle changes in the sequence and length of peptides representing the central and C-terminal part of the HIV-2 V3 region resulted in major differences in the ability to elicit neutralizing antibodies in guinea pigs. One probable explanation for this is that the
Acknowledgments
We thank Mariethe Ehnlund for excellent technical assistance and Johan Rosenqvist for peptide immunization of guinea pigs. This work was supported by the Swedish Agency for Research Cooperation and Swedish Physicians Against AIDS.
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