The expression of HIV-1 coreceptors (CXCR4 and CCR5) on monocyte surface can be regulated by the ligand of CD14 (LPS), which stimulate the susceptibility of the cells to HIV-1. To investigate whether it exists potential association between CD14 and HIV-1 coreceptor CXCR4, we tested the impact of CD14-specific monoclonal antibodies (mAbs) upon CXCR4-dependent responses, such as SDF-induced chemotaxis and HIV Env-mediated membrane fusion. The anti-CD14 mAb TUK4 like CXCR4-specific mAb 12G5 could block SDF-induced chemotaxis of U937 cells in a dose-dependent manner, while another CD14-specific mAb UCHM-1 did not show any activity. More interestingly, syncytium assay indicated that only the CD14-specific mAb TUK4 inhibited HIV Env-mediated CXCR4-dependent cell fusion between U937 cells and HIV-1HXB2 Env transfected CHO cells distinctly, consistent with its activity against CXCR4-dependent chemotaxis. These results provided experimental evidence for existence of close association between CD14 and HIV coreceptor CXCR4 on human monocytic cells.

Some neutralizing epitopes on HIV-1 envelope proteins were shown to induce antibodies that could effectively inhibit the infection of different HIV-1 strains in vitro) But only very low levels of antibodies to these epitopes were determined in the HIV-1 infected individuals) In this study, the aluminum(alum)adjuvant to increase the immunogenicity of the neutralizing epitopes was used) Three epitope-peptides wC-(ELDKWAG)4, C-(RILAVERYLKD-G)2and C-(GPGRAFY)2 x, which contain three epitopes(ELDKWA, RILAVERYLKD, GPGRAFY)from the HIV-1 Env proteins, were synthesized and conjugated to carrier protein keyhole limpet hemocyanin(KLH)) The epitope-vaccines C-(ELDKWAG)4-KLH and C(RILAVERYLKD-G)2-KLH in alum induced high levels of epitope-specific antibodies recognizing the epitopes from epitope-peptides C-(ELDKWAG)4and C-(RILAVERYLKD-G)2, as well as the gp41 C-domain peptides P2 wCTSLIHSLIEESQNQQEKNEQELLELDKWA(aa 646-674)xand P1wLQARILAVERYLKDQQL(aa 583-599)x and the recombinant soluble gp41(rsgp41)bearing both epitopes(antibody titer in rabbit sera was 1:12800-25,600 dilution)) Immunoblotting analysis demonstrated that the antibodies in both antisera bound to rsgp41, indicating that both antibodies recognized the natural epitopes on rsgp41 protein) The epitope-vaccines C-(GPGRAFY)2-KLH induced moderate GPGRAFY epitope-specific antibody response with a titer of 1:6400) In contrast, as it was demonstrated in previous studies, the immunization with rgp160 induced weak antibody response to these three epitopes(titer of 1:400-1600)) This suggests that E pitope-peptides conjugated to KLH when infected with alum significantly increases immunogenicity of gp41 neutralizing epitopes providing a hope for the development of an HIV-1 vaccine)

An effective vaccine is urgently needed to stop AIDS-epidemic. Up to now none of the candidate HIV-vaccines has been developed to prevent HIV-1 infection. A few neutralizing antibodies against HIV-1 enveloping proteins proved to be highly effective to neutralize different strains in vitro. Unfortunately, these antibodies are rare in infected humans, and have never yet been raised by a vaccine. The multiple sequential and antigenic variability of HIV-1 led to unprecedented difficulties in development of effective vaccines and anti-viral drugs. More and more experimental evidences indicated that HIV-1 mutants resulted in immune evasion may be a grave challenge for conventional strategy to prepare effective vaccines. We suggested that epitope-vaccine could be a new strategy to induce high levels of neutralizing antibodies with predefined epitope-specificity against HIV-1. Several candidate epitope-vaccines including mono-epitope-vaccine, multi-epitope-vaccine, epitope-vaccines in combination, were prepared and systematically studied in animal experiments. These studies provided experimental evidences that epitope-vaccine could be a new strategy to develop effective vaccines for prevention and immunotherapy against viral infection of HIV-l or other viruses.

nd human Iymphocytes, a highly conserved region (aa583-599; LQARILAV-ERYLKDQQL) was identified in gp41 as a binding site for human T cells and monocytes1-3, Gp41 could thus inhibit lym-phocyte proliferation4 and upregulate major histocompatibility complex (MHC) class I, class II and intercellular adhesion molecule 1 (ICAM-1) expression5-effects similar to those of human type I interferons. When we compared the sequences of gp41 and type I interferons, a similar epi-tope RILAV--YLKD was found in the im-munosuppressive domain (ISD) of gp41 and two regions in interferon α(IFN-α) (aa29-35 and 113-129), IFN-β(aa31-27 and 125-138) and IFN-ω(aa29-35 and 123-136) (Fig.1). Surprisingly, the common epitope exists within the receptor binding sites of gp41, IFN-α and IFN-β(Ref.6.) We characterized the common epitope and found increased levels of antibodies to IFN-αand IFN-βin20 HIV-infectedindividuals, compared with levels in normal healthy controls; these lev-els subsequently decreased with disease progression. Affinity-purified anti--gp41 an-tibody from HIV-infected individduals was shown to recognize human IFN-αand IFN-β. In addition, anti-IFN-αand IFN-β antibodies interacted with gp41 and its peptide(aa583-599), and inhibited gp41 binding to human lymphocytes. These results suggest that the common immunological epitope within gp41 induces incrased levels of an-tibodies that can interact with IFN-αand IFN-β. Gringeri et al. demonstrated that HIV-infected patients treated with an IFN-α vac-cine showed a significant reduction in the rate of disease progression, which was asso-ciated with an increase in IFN-αantibody titer and an increase in IFN-α-neutralizing capacity. The anti-IFN-α antibody was shown to recognize aa560-599 of HIV-1 gp41 (Ref. 7). These data suggest that the common immunological epitope within re-ceptor binding sites may be associated with protetive activity. Interestingly, a similar epitope in SIV gp32 could protect macaques from SIV infection89, indication that the common immunological epitope on gp41, IFN-αand IFN-β could be useful in the de-velopment of a vaccine strategy against HIV-infection.

It is generally agreed that the recombinant gp120 (rgp120) of HIV-1 could be developed as an antibody-mediated subunit vaccine against HIV-1. Unfortunately, Connor and co-workers have provided direct experimental evidence that rgp120 (monomer) does not protect individuals from HIV-1 infection1. In addition, Moore has reviewed studies of rgp120 subunit vaccines and proposed that a new strategy is needed to develop an effective vaccine against HIV-1 (Ref. 2). An epitope vaccine might be such a strategy. The epitope vaccine against HIV-1 uses much of the principal neutralizing determinant (PND) of the envelope (Env) protein (gp160) and belongs to a special type of synthetic peptide vaccine. Several epitopes on gp160 have been characterized as PNDs, for example, GPGRAF (Env aa316-321) on the V3 loop of gp120, ELDKWA (aa669-674) on the C-domain of gp41 and RILAVERYLKD (Env aa586-596) on the N-domain of gp41. It has been demonstrated that crossneutralizing antibodies elicited by peptides of the V3 loop bind to epitope GPGRAF, which undergoes restricted mutation from GPGRAF to GPG(R/K/Q)AF (Ref. 3). ELDKWA is a relatively conserved epitope. Recent sequence analysis of primary isolates from different HIV-1 subtypes suggests that the major determinant of monoclonal antibody (mAb) 2F5 corresponds to the amino acid sequence LDKW. Naturally occurring and in vitro-selected neutralization-resistant viruses contained D to N, D to E and K to N changes in the ELDKWA motif. These amino acid changes caused abrogation of 2F5-binding to ELDKWA (Ref. 4). Recent studies have attested the breadth of reactivity of 2F5 by the antibody's significant neutralization potency against African, Asian, American and European strains from clades A, B and E. Most of the viruses investigated were neutralized by 90% (Ref. 4). The epitope RILAVERYLKD has been shown to induce protective activity5. We have designed candidate synthetic peptide vaccines and compared immunogenicity of: (1) the recombinant gp160 and gp41 subunit vaccines; (2) peptide vaccines of the C-domain on gp41 (EnvIIIB aa646-674: C-TSLIHSLIEESQNQQEKNEQELLELDKWA) and the gp120 V3 loop (EnvIIIB aa301-328: C-TRPNNNTRKSIRIQRGPGRAFYTIGKI); and (3) epitope vaccines of ELDKWA on the C-domain of gp41 [C-(ELDKWAG)4-BSA] and GPGRAFY on gp120 [C-(GPGRAFY)4-P24EC]. In mice, the rgp160 subunit vaccine induced a very weak antibody response to both epitopes (ELDKWA and GPGRAFY), whereas both synthetic peptides conjugated with carrier protein BSA or carrier peptide P24EC (GPKEPFRDYVDRFYK-C) increased the epitope-specific antibody responses (increased antibody titre by two- to fourfold) to ELDKWA and GPGRAFY. Interestingly, both the ELDKWA and the GPGRAFY tetramer epitope vaccines induced a strong epitope-specific antibody response against ELDKWA and GPGRAFY, respectively, and the levels of ELDKWA- and GPGRAFY-specific antibodies increased two- to fourfold, compared with levels induced with the synthetic peptide vaccines (Table 1). These results indicate that the epitope vaccine could provide a new strategy to develop an effective vaccine against HIV-1 infection. Crystallographic analysis indicates that the binding of gp120 to both CD4 and a chemokine receptor induces conformational changes in gp41 from the native structure to the fusion-active structure. The exposed coiled coils create a fusion intermediate, in which the C-domain and N-domain are exposed6. Recent studies have demonstrated that a fusion-competent vaccine with broad neutralization of HIV primary isolates is associated with these fusion intermediates7,8, suggesting that