Former investigations demonstrated that the envelope glycoprotein E2 could protect pigs from classical swine fever virus (CSFV). Based on these findings, we prepared synthetic peptide vaccine using E2 N-terminal antigenic units B/C and hoped to induce protective activity against lethal challenge of virulent CSFV strain Shimen. Five overlapped peptides sequence-covering amino acids 693-777 on E2 of Shimen were synthesized and then conjugated with bovine serum albumin (BSA), respectively. In the vaccination course, the candidate peptide vaccines in combination (multi-peptide vaccine (MPV)) were applied for immunization of pigs (n = 10) and induced strong antibody response against CSFV. It is subsequently demonstrated that this peptide vaccine could provide immunized pigs complete protection against lethal CSFV challenge as C-strain does, while all non-immunized pigs in negative control group manifested obvious typical symptoms and died during the second and third weeks after viral challenge. In order to confirm the neutralizing activity of the polyclonal antibodies induced by MPV, neutralization assay were carried out on rabbits. The live C-strain alone could ordinarily induce typical fever on rabbits. The typical fever of rabbits induced by the live C-strain could be inhibited by pre-incubation with the anti-sera (dilution 1:4 and 1:16) induced by MPV, but not inhibited by pre-incubation with the same anti-sera from which the antibodies against five peptides were removed by peptide-specific affinity chromatography, which indicates that these peptide-specific antibodies in the anti-sera induced by MPV provided protective activity against CSFV. Our finding provides a new way to develop marker vaccine against CSFV.

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.

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)

Recent studies demonstrated that the N- and C-domains of HIV-1 gp41 is involved in virus-mediated membrane fusion resulting in HIV-entry into the target cells. Up to now, viral mutation baffled many scientists to develop effective vaccines and drugs against HIV-1. To acquire more information of mutation of gp41 and to reveal the relationship of structure and function of the N- and C-domains, we compared and analyzed amino acid sequences of the gp41 ectodomain (aa 512-681) of 862 isolates from most HIV-1 clades (including A, B, C, D, E, F, G, H, I, J and O clades). A consensus sequence of the ectodomain with the highest frequency emerging on each position is constituted. The fusion domain and the N-domain belong to the most conserved regions in gp41, and most variable residues assemble partial to the C terminal of gp41. The hydrophobicity of each position is also calculated. The a and d positions in the N-domain for maintaining stabilization of the trimeric coiled coil interactions are highly conservative, and the e and g positions in the C-domain to retain the interaction show also highly conservative. The strange high conservation of the c residues may have an implication in the coiled coil structure. The highly conserved residues form the lining of the hydrophobic cavity and the deep cavity is an ideal target for small molecular inhibitors. On the C-terminal of the C-domain there is a highly conserved segment GIVQQQ. They are intimately involved in forming the three interfaces between neighboring helices. The function of the N- and C-domains, such as binding to the potential cellular receptor and inducing protective activities, are also discussed. These studies on the mutation, structure and functions of the N-and C-domains suggested that both domains become a new focus to develop effective vaccine and antiviral drugs in the new strategies.

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.