In summary, our study describes the potential of oral vaccines with replication-attenuated viral vectors for repeated induction and augmentation of HIV Env-specific cellular and humoral responses

In summary, our study describes the potential of oral vaccines with replication-attenuated viral vectors for repeated induction and augmentation of HIV Env-specific cellular and humoral responses. mucosal cytotoxic T-lymphocyte response by intrarectal immunization with MVA expressing the HIV Env glycoprotein and indicated that this non-replicating recombinant MVA may be at least as effective for mucosal immunization as replicating rVV. A number of more-attenuated VV-derived strains, including MVA (Mayr and and elicited Env-specific immune responses in mucosal and systemic tissues Rabbit Polyclonal to TALL-2 of BALB/c mice after repeated oral delivery. These results suggest that the MVAIIIB/-gal vector can be modified for effective shielding against pre-existing poxvirus neutralizing antibodies for multiple deliveries. METHODS Reagents TMPEG glycol and PEG were obtained from Sigma. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-neutralization assays Neutralization assays were performed using baby hamster kidney cells (BHK-21; ATCC) and pooled sera from mice immunized four times with the WR strain of non-recombinant VV with an end-point antibody titre against VV of 1 1 : 10 000. For infection, free MVAIIIB/-gal or liposome-complexed MVAIIIB/-gal (10 p.f.u. ml?1) was combined with pooled immune or control sera (diluted 1 : 10) for 1 h. The mixtures were then added to a monolayer of BHK cells and incubated for an additional 24 h. The cells were lysed and the level of -gal was measured using a -gal reporter gene activity detection kit (Sigma) Zatebradine hydrochloride according to the manufacturers instructions. All experiments were performed in triplicate. Analyses of fluorescently labelled MVAIIIB/-galCTMPEG/liposome complexes Zatebradine hydrochloride in infected cells MVAIIIB/-gal (107 p.f.u.) was labelled with 4 l DiI (Vybrant Cell-labelling Solutions; Molecular Probes), washed twice with PBS by centrifugation at 30 000 g for 50 min and used for infection of Zatebradine hydrochloride BHK-21 cells in the presence or absence of neutralizing antibodies. In parallel, a liposome film was prepared containing 4 l DiO dye (Molecular Probes). The DiO-labelled liposomes were washed twice with PBS using a stirred ultrafiltration cell (Millipore) and combined with DiI-labelled MVAIIIB/-gal for infection of BHK-21 cells. After 15 or 30 min of infection, the cells were washed three times with PBS, fixed with 3.7% paraformaldehyde and mounted for immunofluorescence analyses. The functional integrity of DiI- and DiO-labelled MVAIIIB/galCTMPEG/liposome complexes was analysed by confocal microscopy 30 min after infection, as described previously (Gaidarov by measuring the ability of the serially diluted sera from the WR VV-immunized mice to inhibit MVAIIIB/-gal infection of BHK-21 cells. Immunized mice (delivery to elicit optimal levels of immune responses. The induction of Env-specific IFN- responses in CD8+ T lymphocytes in spleen and Peyers patches was analysed after each immunization by ELISPOT using a CD8+ T-cell peptide epitope of the Env glycoprotein, I10. As shown in Fig. 4(a), Env-specific IFN- responses in spleen were more than twofold higher after the second immunization in all groups of immunized mice, consistent with the undetectable level of neutralizing antibody responses to MVAIIIB/-gal after the first immunization.Mice that were immunized with the TMPEG and MVAIIIB/-gal preparations, delivered separately or as MVAIIIB/-galCTMPEG/liposome complexes, had similar responses to those induced by the MVAIIIB/-gal virus, suggesting that this preparation of TMPEG/liposomes had no adjuvant effect on the level of Env-specific IFN- production or the neutralizing antibody response to the viral vector. The profile of the IFN- response in Peyers patches was similar to that measured in the spleens of immunized mice, although the frequencies of IFN–secreting CD8+ T cells were lower after the first and second immunizations compared with those in splenocytes of the immunized mice. The level of l10 peptide-specific IFN- responses in spleen and Peyers patches decreased after the third vaccination in mice immunized with the free MVAIIIB/-gal virus or MVAIIIB/-gal and TMPEG/liposomes delivered separately. This could be related to the loss of some Env-specific CD8+effector T cells induced by the first and second immunizations and the ineffective delivery of the env gene by MVAIIIB/-gal due to generation of neutralizing antibodies that interfered with the infection process. Although we were unable to demonstrate any neutralizing antibodies in faecal washes from immunized mice due to the faecal material being diluted extensively, neutralizing antibodies were detectable after the second immunization at serumdilutions lower than 1 : 10. In contrast, the numbers of IFN- producing CD8+ T lymphocytes in mice immunized with the MVAIIIB/-galCTMPEG/liposome complexes remained relatively stable after the third and.