Stem Cells 2005;23:355-364

We previously established a nanosized nasal vaccine delivery system by using a cationic cholesteryl group-bearing pullulan nanogel (cCHP nanogel), which is a universal protein-based antigen-delivery vehicle for adjuvant-free nasal vaccination. In the present study, we examined the central nervous system safety and efficacy of nasal vaccination with our developed cCHP nanogel containing pneumococcal surface protein A (PspA-nanogel) against pneumococcal infection in nonhuman primates. When [18F]-labeled PspA-nanogel was nasally administered to a rhesus macaque (Macaca mulatta), longer-term retention of PspA was noted in the nasal cavity when compared with administration of PspA alone. Of importance, no deposition of [18F]-PspA was seen in the olfactory bulbs or brain. Nasal PspA-nanogel vaccination effectively induced PspA-specific serum IgG with protective activity and mucosal secretory IgA (SIgA) Ab responses in cynomolgus macaques (Macaca fascicularis). Nasal PspA-nanogel-induced immune responses were mediated through T-helper (Th) 2 and Th17 cytokine responses concomitantly with marked increases in the levels of miR-181a and miR-326 in the serum and respiratory tract tissues, respectively, of the macaques. These results demonstrate that nasal PspA-nanogel vaccination is a safe and effective strategy for the development of a nasal vaccine for the prevention of pneumonia in humans.

SummaryPlants have been used as expression systems for a number of vaccines. However, the expression of vaccines in plants sometimes results in unexpected modification of the vaccines by N-terminal blocking and sugar-chain attachment. Although MucoRice-CTB was thought to be the first cold-chain-free and unpurified oral vaccine, the molecular heterogeneity of MucoRice-CTB, together with plant-based sugar modifications of the CTB protein, has made it difficult to assess immunological activity of vaccine and yield from rice seed. Using a T-DNA vector driven by a prolamin promoter and a signal peptide added to an overexpression vaccine cassette, we established MucoRice-CTB/Q as a new generation oral cholera vaccine for humans use. We confirmed that MucoRice-CTB/Q produces a single CTB monomer with an Asn to Gln substitution at the 4th glycosylation position. The complete amino acid sequence of MucoRice-CTB/Q was determined by MS/MS analysis and the exact amount of expressed CTB was determined by SDS-PAGE densitometric analysis to be an average of 2.35 mg of CTB/g of seed. To compare the immunogenicity of MucoRice-CTB/Q, which has no plant-based glycosylation modifications, with that of the original MucoRice-CTB/N, which is modified with a plant N-glycan, we orally immunized mice and macaques with the two preparations. Similar levels of CTB-specific systemic IgG and mucosal IgA antibodies with toxin-neutralizing activity were induced in mice and macaques orally immunized with MucoRice-CTB/Q or MucoRice-CTB/N. These results show that the molecular uniformed MucoRice-CTB/Q vaccine without plant N-glycan has potential as a safe and efficacious oral vaccine candidate for human use.

We found that most peripheral CD4 cells co-express a low density of CD8 alpha antigen in African green monkeys (AGM). Further, the cell surface expression of CD4 and the expression of CD4 mRNA underwent a decrease when purified CD4CD8low cells were cultured with mitogen and IL-2. These observations suggest that AGM CD4 cells are subject to loss of CD4 expression after lymphocyte activation. Part of the peripheral CD8 fraction exhibited a significant helper activity which suggested the phenotypic conversion in helper T cells from CD4+ to CD4- in vivo. Simian immunodeficiency virus (SIV) grew well in CD4 panning cells following SIV infection. In contrast, CD4CD8low cells were resistant to SIV infection after their conversion to CD4- cells.

Transcriptional activation of gene expression directed by the long terminal repeat (LTR) of HIV-1 requires both the transactivation response element (TAR) and Tat protein. HIV-1 mutants lacking a functional tat gene are not able to proliferate. Here we take a genetic approach to suppress HIV-1 replication based on Tat-dependent production of MazF, an ACA-specific endoribonuclease (mRNA interferase) from Escherichia coli. When induced, MazF is known to cause Bak- and NBK-dependent apoptotic cell death in mammalian cells. We first constructed a retroviral vector, in which the mazF (ACA-less) gene was inserted under the control of the HIV-1 LTR, which was then transduced into CD4+ T-lymphoid CEM-SS cells in such a way that, upon HIV-1 infection, the mazF gene is induced to destroy the infecting HIV-1 mRNA, preventing HIV-1 replication. Indeed, when the transduced cells were infected with HIV-1 IIIB, the viral replication was effectively inhibited, as HIV-1 IIIB p24 could not be detected in the culture medium. Consistently, not only cell growth but also the CD4 level was not affected by the infection. These results suggest that the HIV-1-LTR-regulated mazF gene was effectively induced upon HIV-1 IIIB infection, which is sufficient enough to destroy the viral mRNA from the infected HIV-1 IIIB to completely block viral proliferation in the cells, but not to affect normal cell growth. These results indicate that the T cells transduced with the HIV-1-LTR-regulated mazF gene acquire HIV-1 resistance, providing an intriguing potential for the use of the HIV-1-LTR-regulated mazF gene in anti-HIV gene therapy.

Long-term hematopoietic microchimerism from primate ES cells was observed after in vitro differentiation to mesodermal cells, followed by in vivo introduction into the fetal liver microenvironment. The mechanism of such directed differentiation of ES cells remains to be elucidated, but this procedure should allow further investigation.

We have previously reported that CD4/CD8 double-positive (DP) T cells with the resting memory phenotype are present in the periphery of healthy cynomolgus monkeys. In the present study, we performed functional studies on the T cells. The expression of CD4 and CD8 on DP, CD4 single-positive (SP) or CD8 SP T cells was stable in cultures with either mitogen or anti-CD3 antibody stimulation. In spite of lacking CD28 expression, DP T cells showed similar proliferative ability and apoptosis sensitivity to CD4 SP and CD8 SP T cells. DP T cells showed both helper and cytotoxic activities. Although the helper activity of DP T cells was lower than that of CD4 SP T cells, cytotoxic activity was comparable to that of CD8 SP T cells. Fresh DP T cells killed target cells mainly by the perforin-granzyme pathway. In addition, fresh DP T cells expressed a high level of mRNA for IFN-gamma and produced a high level of IFN-gamma when they were activated by anti-CD3 antibody ligation. On the other hand, several expanded DP T cell clones shared TCR V(beta) with expanded CD4 SP T cell clones, strongly suggesting that those two corresponding clones with DP and CD4 SP phenotypes might be derived from the same ancestor T cell. These results showed that the DP T cells are a novel T cell subset with functions overlapping with those of CD4 SP and CD8 SP T cells, and that they might play protective and regulatory roles in secondary immune response in cynomolgus monkeys.

We previously showed that oral immunization of mice with a rice-based vaccine expressing cholera toxin (CT) B subunit (MucoRice-CT-B) induced CT-specific immune responses with toxin-neutralizing activity in both systemic and mucosal compartments. In this study, we examined whether the vaccine can induce CT-specific Ab responses in nonhuman primates. Orally administered MucoRice-CT-B induced high levels of CT-neutralizing serum IgG Abs in the three cynomolgus macaques we immunized. Although the Ab level gradually decreased, detectable levels were maintained for at least 6 mo, and high titers were rapidly recovered after an oral booster dose of the rice-based vaccine. In contrast, no serum IgE Abs against rice storage protein were induced even after multiple immunizations. Additionally, before immunization the macaques harbored intestinal secretory IgA (SIgA) Abs that reacted with both CT and homologous heat-labile enterotoxin produced by enterotoxigenic Escherichia coli and had toxin-neutralizing activity. The SIgA Abs were present in macaques 1 mo to 29 years old, and the level was not enhanced after oral vaccination with MucoRice-CT-B or after subsequent oral administration of the native form of CT. These results show that oral MucoRice-CT-B can effectively induce CT-specific, neutralizing, serum IgG Ab responses even in the presence of pre-existing CT- and heat-labile enterotoxin-reactive intestinal SIgA Abs in nonhuman primates.