Rabies virus (RV) vaccine strain-based vectors show great promise as vaccines against other viral diseases such as human immunodeficiency virus type 1 (HIV-1) infection and hepatitis C, but a low residual pathogenicity remains a concern for their use. Here we describe several highly attenuated second-generation RV-based vaccine vehicles expressing HIV-1 Gag. For this approach, we modified the previously described RV vaccine vector SPBN by replacing the arginine at position 333 (R333) within the RV glycoprotein (G) with glutamic acid (E333), deleting 43 amino acids of the RV G cytoplasmic domain (CD), or combining the R333 exchange and the CD deletion. In addition, we constructed a new RV vector that expresses HIV-1 Gag from an RV transcription unit upstream of the RV phosphoprotein gene (BNSP-Gag) instead of upstream of the G gene. As expected and as demonstrated for SPBN-Gag, all vaccine vehicles were apathogenic after peripheral administration. However, the new, second-generation vaccine vectors containing modifications in the RV G were also apathogenic after intracranial infection with 10 5 infectious particles, and BNSP-Gag produced a 50%-reduced mortality in mice. Of note, the observed attenuation of pathogenicity did not result in either the attenuation of the humoral response against the RV G or the previously observed robust cellular response against HIV-1 Gag. These findings demonstrate that very safe and highly effective RV-based vaccines can be constructed and further emphasize their potential utility as efficacious antiviral vaccines.Rabies virus (RV) is a nonsegmented negative-strand RNA virus of the Rhabdoviridae family. The 12-kb RV genome encodes five monocistronic RNAs encoding the nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), the single transmembrane protein G, and the viral polymerase (L). The RV virion consists of an internal core or ribonucleoprotein (RNP) complex, which is composed of the viral RNA encased in the N protein and associated with P and L (32) and an external component, the viral envelope, which consists of the host-cell derived membrane with the M protein located at its inner surface and the membrane-spanning G protein (18,19). The G protein is responsible for both the interaction with a cellular receptor(s) and pH-dependent membrane fusion (33).RV pathogenicity has been studied for more than 100 years, with research results indicating that RV consists of a wide array of variants. These can range from highly pathogenic strains, such as silver-haired bat virus, to extremely attenuated RV vaccine strains, such as SAG-2, which are not pathogenic in severe combined immunodeficiency (SCID) mice after oral application (20, 23; C. Hanlon, M. Fiorello, C. L. Schumacher, V. Shankar, A. Hamir, and C. Rupprecht, Abstr. 4th Annu. Int. Meet. Adv. Rabies Control Americas, 1993). Two proteins have been associated with RV pathogenesis, namely, P and G. It has been suggested that a specific interaction of a conserved domain within RV P and the cytoplasmic dynein light chain ...

In this study, we suggest that CD8 levels on T cells are not static, but can change and, as a result, modulate CD8+ T cell responses. We describe three models of CD8 modulation using novel weak-agonist (K1A) and super-agonist (C2A) altered peptide ligands of the HY smcy peptide. First, we used peripheral nonresponsive CD8low T cells produced after peripheral HY-Db MHC class I tetramer stimulation of female HY TCR transgenic and wild-type mice. Second, we used genetically lowered CD8int T cells from heterozygote CD8+/0 mice. Finally, we used pre-existing nonresponsive CD8low T cells from male HY TCR transgenic mice. In CD8low and CD8high mice, presence of a lower level of CD8 greatly decreased the avidity of the peptide-MHC for HY TCR as reflected by avidity (KD) and dissociation constant (T1/2) measurements. All three models demonstrated that lowering CD8 levels resulted in the requirement for a higher avidity peptide-MHC interaction with the TCR to respond equivalently to unmanipulated CD8high T cells of the same specificity. Additionally, direct injections of wild-type HY-Db and C2A-Db tetramers into female HY TCR or female B6 mice induced a high frequency of peripheral nonresponsive CD8low T cells, yet C2A-Db was superior in inducing a primed CD8+CD44+ memory population. The ability to dynamically modulate the size and responsiveness of an Ag-specific T cell pool by “CD8 tuning” of the T cell during the early phases of an immune response has important implications for the balance of responsiveness, memory, and tolerance.

Recombinant, replication-competent rabies virus (RV) vaccine strain-based vectors were developed expressing HIV type I (HIV-1) envelope glycoprotein (gp160) from both a laboratory-adapted (CXCR4-tropic) and a primary (dual-tropic) HIV-1 isolate. An additional transcription stop/start unit within the RV genome was used to express HIV-1 gp160 in addition to the other RV proteins. The HIV-1 gp160 protein was stably and functionally expressed, as indicated by fusion of human T cell lines after infection with the recombinant RVs. Inoculation of mice with the recombinant RVs expressing HIV-1 gp160 induced a strong humoral response directed against the HIV-1 envelope protein after a single boost with recombinant HIV-1 gp120 protein. Moreover, high neutralization titers up to 1:800 against HIV-1 could be detected in the mouse sera. These data indicate that a live recombinant RV, a rhabdovirus, expressing HIV-1 gp160 may serve as an effective vector for an HIV-1 vaccine.

A highly attenuated, recombinant rabies virus (RV) vaccine strain-based vector was utilized as a new immunization strategy to induce humoral and cellular responses against hepatitis C (HCV) glycoprotein E2. We showed previously that RV-based vectors are able to induce strong immune responses against human immunodeficiency virus type I (HIV-1) antigens. Here we constructed and characterized three replication-competent RV-based vectors expressing either both HCV envelope proteins E1 and E2 or a modified version of E2 which lacks 85 amino acids of its carboxy terminus and contains the human CD4 transmembrane domain and the CD4 or RV glycoprotein cytoplasmic domain. All three constructs stably expressed the respective protein(s) as indicated by Western blotting and immunostaining. Moreover, surface expression of HCV E2 resulted in efficient incorporation of the HCV envelope protein regardless of the presence of the RV G cytoplasmic domain, which was described previously as a requirement for incorporation of foreign glycoproteins into RV particles. Killed and purified RV virions containing HCV E2 were highly immunogenic in mice and also proved useful as a diagnostic tool, as indicated by a specific reaction with sera from HCV-infected patients. In addition, RV vaccine vehicles were able to induce cellular responses against HCV E2. These results further suggest that recombinant RVs are potentially useful vaccine vectors against important human viral diseases.

Live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. However, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. Using the rabies virus (RV) envelope protein as a carrier molecule, we show here that inactivated RV particles can be utilized to present Bacillus anthracis protective antigen (PA) domain-4 in the viral membrane. In addition to the RV glycoprotein (G) transmembrane and cytoplasmic domains, a portion of the RV G ectodomain was required to express the chimeric RV G anthrax PA on the cell surface. The novel antigen was also efficiently incorporated into RV virions. Mice immunized with the inactivated recombinant RV virions exhibited seroconversion against both RV G and anthrax PA, and a second inoculation greatly increased these responses. These data demonstrate that a viral envelope protein can carry a bacterial protein and that a viral carrier can display whole polypeptides compared to the limited epitope presentation of previous viral systems.