Caprine Arthritis Encephalitis Virus (CAEV) is a lentivirus closely related to visna virus of sheep and more distantly related to the human lentivirus HIV-1. The genomes of lentiviruses contain additional genes that regulate the lentivirus gene expression; one of these is Rev, a protein that regulates the expression of viral proteins via post-transcriptional mechanisms. A cDNA clone was isolated from CAEV infected cells and shown to encode the 18-kDa Rev protein of CAEV. Antibodies against CAEV Rev (Rev-C) demonstrated that the CAEV Ray protein accumulated in the nucleus and in particular in the nucleolus of transiently transfected cells. Mutation of a basic region in the CAEV Rev protein resulted in loss of nucleolar localization. A highly structured RNA element has been identified in the env gene of CAEV (nt 7850-8150); its structure and location suggested that it was analogous to the Ray-responsive element (RRE) of HIV-1 and visna virus. A 300-bp fragment (nt 7850-8150) spanning this region was substituted for the HIV-1 RRE in an HIV-1 Gag expression vector. Expression of the Gag protein was dramatically increased when Rev-C was added in trans, indicating that this fragment contained the cis-acting CAEV Rev Responsive Element. Cross-activation by the Rev/Rex proteins of other lentiviruses and members of the HTLV-I family indicated that this RRE could interact with Rev or Rex proteins of other viruses. This suggests that the highly divergent lentiviruses share similar mechanisms and cofactors regulating post-transcriptional viral gene expression. The Rev/RRE mechanism is thus the most conserved regulatory mechanism in lentiviruses and other complex retroviruses.
Results DNA delivery using EP led to greatly enhanced expression of SIV antigens and increased cellular responses (up 3% of total T cells) that were broad, long-lasting (up to 45 weeks post vaccination) and included cells of multifunctional phenotype. SIV-specific cellular responses were found both in blood, BAL and rectal mucosa. Interestingly, compared to blood, the responses in BAL were consistently higher (up to 2 log) and included a higher frequency of polyfunctional cells. The cellular responses were characterized as predominantly EM CD4+ and CD8+ T cells in BAL and as both CM and EM T cells in blood. EP DNA delivery elicited both systemic and mucosal humoral immune responses, including the induction of Gag-specific IgA. Upon high dose SIVmac251 challenge, the vaccinated animals showed statistically significant lower VL peak in acute (1 log10) and in chronic viremia (1.7 log10).
Background We have previously reported that potent, long-lasting HIV-1 Env-specific cell-mediated immune responses could be elicited in rhesus macaques and mice using plasmids encoding env DNA as the immunogen. Subsequent experiments showed that combination of DNA and protein in the form of inactivated virus particles provided significant protection from infection and high viremia. We examine a vaccine platform combining DNA and recombinant Env protein co-immunization at the same time to generate both strong cellular and humoral immune responses. Methods
Methods 250 HIV-1 clade B infected individuals were screened for T cell responses to the entire HIV proteome. This yielded 26 regions in HIV-1 Gag, Pol, Vif and Nef proteins that were i) preferentially targeted by individuals with low viral loads, ii) more conserved and iii) elicited responses of higher functional avidity and broader cross-reactivity than responses to other, less-beneficial regions. The ‘beneficial’ segments were linked by triple alanines, translated into an expression-optimized nucleotide sequence and cloned into a CMV plasmid harboring a GM-CSF signal peptide. Immunogenicity was evaluated in C57BL/6 mice two weeks after a second DNA vaccination. Cellular immune responses were characterized using intracellular cytokine staining and IFN-g ELISPOT using overlapping peptide pools covering the segments included in the T-cell immunogen. Results Vaccination with 20 μg of DNA generated both CD4 and CD8 IFN-g+ responses to the immunogen sequence. The T-cell immunogen elicited a more balanced, broad T cell response to all protein components (Gag, Pol, Vif and Nef) contained in the immunogen than immunizations using plasmids encoding for the entire Gag, Pol, Nef, Tat and Vif proteins, which induced a strong Gag dominance.
Background Immunodominance and sequence diversity are major hurdles in the development of effective HIV vaccines. We tested the hypothesis that a vaccine candidate composed of strictly Conserved Elements (CE) of the HIV proteome excluding the variable regions would help overcome problems of viral sequence diversity and potential negative effects of immunodominance. Seven CE were identified in p24. Vaccination of macaques with p55DNA failed to elicit cellular or humoral immune responses to the CE, while epitopes outside of the CE were immunogenic.
Background We have developed a vaccine design that utilizes the unique property of the endoplasmic reticulum chaperon, heat shock protein (HSP) gp96, to bind antigenic peptides and deliver them to APCs. Cell-based gp96-Ig vaccines, by prolonged in vivo secretion of gp96-Ig peptide, imitate viral replication and provide immune stimuli comparable to attenuated viruses. In model systems in mice we have shown that gp96-Ig transfected, antigen expressing cells secrete gp96-Ig in vivo and stimulate both systemic and strong mucosal immunity. The aim of our study was to evaluate safety and systemic and mucosal SIV-immunity with secreted gp96-Ig-SIV vaccines in non-human primates.
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