Purified herpes simplex type 1 glycoprotein D (gD) genetically fused with the type 16 human papillomavirus E7 oncoprotein enhances antigen-specific CD8+ T cell responses and confers protective antitumor immunity.

Type 1 herpes virus (HSV-1) glycoprotein D (gD) enhances antigen-specific immune responses, particularly CD8(+) T cell responses, in mice immunized with DNA vaccines encoding hybrid proteins genetically fused with the target antigen at a site near the C-terminal end. These effects are attributed to the interaction of gD with the herpes virus entry mediator (HVEM) and the concomitant blockade of a coinhibitory mechanism mediated by the B- and T-lymphocyte attenuator (BTLA). However, questions concerning the requirement for endogenous synthesis of the antigen or the adjuvant/antigen fusion itself have not been addressed so far. In the present study, we investigated these points using purified recombinant gDs, genetically fused or not with type 16 papilloma virus (HPV-16) E7 oncoprotein. Soluble recombinant gDs, but not denatured forms, retained the ability to bind surface-exposed cellular receptors of HVEM-expressing U937 cells. In addition, in vivo administration of the recombinant proteins, particularly gD genetically fused with E7 (gDE7), promoted the activation of dendritic cells (DC) and antigen-specific cytotoxic CD8(+) T cells. More relevantly, mice immunized with the gDE7 protein developed complete preventive and partial therapeutic antitumor protection, as measured in mice following the implantation of TC-1 cells expressing HPV-16 oncoproteins. Collectively, these results demonstrate that the T cell adjuvant effects of the HSV-1 gD protein did not require endogenous synthesis and could be demonstrated in mice immunized with purified recombinant proteins.

Ratio of HCV structural antigens in protein-based vaccine formulations is critical for functional immune response induction.

HCV (hepatitis C virus) infection is among the leading causes of chronic liver disease, but currently there is no vaccine available. Data have accumulated about the importance of targeting different HCV antigens in vaccine candidate preparations. Here, a surface response study to select the optimal ratio of recombinant HCV structural antigens in a vaccine preparation, capable of generating in vivo functional cellular immune response in mice, was performed. The immunogenicity of the selected HCV structural protein mixture (Co-E1-E2) in mice and African green monkeys, after five doses of immunization, was also demonstrated. Specific T-cell proliferative response against HCV structural antigens was induced in vaccinated mice. Moreover, on challenge with recombinant HCV VV (vaccinia virus), all mice controlled the viraemia and 80% were protected. On the other hand, monkeys immunized with Co-E1-E2 developed antibodies, specifically directed to region 412-438 of E2 protein, that include an epitope implicated in HCV neutralization, in addition to a specific proliferative response against HCV Core and E2 proteins. These results indicated that the optimal amount and ratio of HCV recombinant proteins should be taken into account to elicit a successful immune response against HCV and therefore have important implications for vaccine design.

Intranasal immunization with a proteoliposome-derived cochleate containing recombinant gD protein confers protective immunity against genital herpes in mice.

The purpose of this study was to investigate the potential of intranasal (IN) immunization with Neisseria meningitides B proteoliposome (AFPL1) and AFPL1-derived cochleate (AFCo1), containing glycoprotein D (gD) of herpes simplex virus type 2 (HSV-2) for induction of protective immunity against genital herpes infection in mice. We could show that IN immunization with both AFPL1 and AFCo1 containing gD induced gD-specific IgG antibody and lymphoproliferative responses. However, IFN-gamma response could only be detected in CD4(+) splenic cells and genital lymph node cells of the AFCo1gD immunized mice upon recall antigen stimulation in vitro. Importantly, IN immunization with AFCo1gD could elicit a complete protection against an otherwise lethal vaginal challenge with HSV-2, while the AFPL1gD immunized mice were only partially protected. Further, we could show that the IFN-gamma response and protective immunity observed after IN immunization with AFCo1gD are mediated via the adaptor molecule myeloid differentiation factor 88. These data may have implications for the development of a mucosal vaccine against genital herpes.

Interleukin-8 mRNA synthesis and protein secretion are continuously up-regulated by respiratory syncytial virus persistently infected cells.

The aim of this study was to investigate whether respiratory syncytial virus persistence regulates interleukin 8 (IL-8) mRNA synthesis and protein secretion in a human lung epithelial cell line (A549). Therefore, we established RSV persistence in these cells (A549per) and determined the levels of interleukin-8 mRNA by RT-PCR and of protein through ELISA. Interleukin-8 mRNA synthesis and protein secretion were continuously up-regulated in A549per cells during passages and in A549 cells that had been incubated with supernatants (cA549per) obtained from A549per passages. These results suggested that the enhancement of interleukin-8 was stimulated either by the presence of the RSV genome in the cell or by soluble mediator(s) induced by RSV, which, in turn, increased interleukin-8 mRNA synthesis and protein secretion. Soluble RSV F and G proteins were identified as mediators. Moreover, interleukin-8 enhancement was observed after 1-min incubation with the soluble mediators, thus suggesting that interleukin-8 up-regulation was triggered by receptor-ligand interaction.

A B-lymphocyte binding peptide from BNRF1 induced antibodies inhibiting EBV-invasion of B-lymphocytes.

Epstein-Barr virus (EBV) infects human target cells mainly through gp350/220-CD21 and gp42-MHCII interactions; however, it has been shown that these interactions are dispensable for EBV-invasion of susceptible cells, suggesting that other viral proteins are involved in this process. It is probable that tegument BNRF1/p140 protein is involved in EBV-invasion of target cells, since anti-p140 antibodies inhibit EBV-infection of B-lymphocytes and there is evidence that part of the protein is located on virus surface. Sixty-six peptides, covering the entire BNRF1/p140 sequence, were synthesised and tested in lymphoblastoid cell line binding assays. Peptides 11465 and 11521 bound with high affinity to Raji, Ramos and P3HR-1 cells but not to erythrocytes, showing cell-binding behaviour similar to EBV. These two peptides induced antibodies recognising live EBV-infected cells. Interestingly, peptide-11521 (YVLQNAHQIACHFHSNGTDA) or antibodies induced by this peptide inhibited EBV-binding to B-lymphocytes, suggesting that this p140-region could be involved in EBV and B-lymphocyte interaction.

Low pH-induced conformational changes in vesicular stomatitis virus glycoprotein involve dramatic structure reorganization.

Membrane fusion is the key step in the entry of enveloped animal viruses into their host cells. Fusion of vesicular stomatitis virus with membranes occurs at acidic pH and is mediated by its envelope glycoprotein, the G protein. To study the structural transitions induced by acidic pH on G protein, we have extracted the protein from purified virus by incubation with nonionic detergent. At pH 6.0, purified G protein was able to mediate fusion of either phospholipid vesicles or Vero cells in culture. Intrinsic fluorescence studies revealed that changes in the environment of Trp residues occurred as pH decreases. In the absence of lipidic membranes, acidification led to G protein aggregation, whereas protein-protein interactions were substituted by protein-lipid interactions in the presence of liposomes. 1,1'-Bis(4-aniline-5-naphthalene sulfonate) (bis-ANS) binding was utilized to probe the degree of exposure of hydrophobic regions of G protein during acidification. Bis-ANS binding was maximal at pH 6.2, suggesting that a hydrophobic segment is exposed to the medium at this pH. At pH 6.0, a dramatic decrease in bis-ANS binding was observed, probably due to loss of tridimensional structure during the conformational rearrangement. This hypothesis was confirmed by circular dichroism analysis at different pH values, which showed a great decrease in alpha-helix content at pH values close to 6.0, suggesting that a reorganization of G protein secondary structure occurs during the fusion reaction. Our results indicate that G protein undergoes dramatic structural changes at acidic pH and acquires a conformational state able to interact with the target membrane.

Localization of a protective epitope on a Venezuelan equine encephalomyelitis (VEE) virus peptide that protects mice from both epizootic and enzootic VEE virus challenge and is immunogenic in horses.

In order to define more precisely the protective epitope encoded within the first 25 amino acids (aa) of the E2 glycoprotein of the Trinidad donkey strain of Venezuelan equine encephalomyelitis (VEE) virus, we examined the immunogenicity of smaller peptides within the first 19 aa. pep1-9 and pep3-10 elicited virus-reactive antibody, but failed to protect mice from virus challenge. Additionally, pep3-10 was identified by a competitive binding assay using overlapping peptide octamers as the putative binding site of the antipeptide monoclonal antibody (mAb) 1A2B-10. Since the E2 amino-terminal sequence for all VEE subtype viruses is conserved, we tested the protective capacity in mice of passively transferred mAb 1A2B-10 and found it to protect from both epizootic and enzootic VEE virus challenge. Since horses are an important natural host for VEE virus, pep1-19 was used to immunize horses and was found to be immunogenic and to elicit virus-reactive antibody.