The GD2 ganglioside expressed on neuroectodermally derived tumors, including neuroblastoma and melanoma, is weakly immunogenic in tumor-bearing patients and induces predominantly immunoglobulin (Ig)-M antibody responses in the immunized host. Here, we investigated whether interconversion of GD2 into a peptide mimetic form would induce GD2 cross-reactive IgG antibody responses in mice. Screening of the X 15 phage display peptide library with the anti-GD2

In this report, we described induction of HIV envelope (env)-specific systemic and mucosal immune responses by oral vaccination of BALB/c mice with env-encoded plasmid DNA encapsulated in poly(dl-lactide-co-glycolide) (PLG) microparticles. We demonstrated that intragastric administration of the encapsulated plasmid DNA resulted in transduced expression of the env glycoprotein in the intestinal epithelium. Mice immunized orally exhibited env-specific type 1 and cytotoxic T lymphocyte (CTL) responses in spleen and the inductive (Peyer's patches) and effector (lamina propria) mucosal tissues of gut. Oral administration of PLG-encapsulated plasmid DNA encoding gp160 also induced env-specific serum antibodies, and an increased level of IgA directed to gp160 was detected in fecal washes of the immunized mice. In contrast, intramuscular (i.m.) administration of naked or PLG-encapsulated DNA vaccine induced only systemic cellular and humoral responses to the env glycoprotein. Using an HIV env-expressing recombinant vaccinia viral intrarectal murine challenge system, we observed higher resistance to mucosal viral transmission in mice immunized orally than in animals injected i.m. with PLG-encapsulated plasmid DNA encoding gp160. Results of these studies demonstrate the feasibility of using orally delivered PLG microparticles containing plasmid DNA-encoded HIV gp160 for induction of env-specific systemic and mucosal immune responses and protection against recombinant HIV env vaccinia virus challenge.

Cytogenetic abnormalities are important diagnostic and prognostic criteria for hematologic malignancies. Karyotyping and fluorescence in situ hybridization (FISH) are the conventional methods by which these abnormalities are detected. The sensitivity of these microscopy‐based methods is limited by the abundance of the abnormal cells in the samples and therefore these analyses are commonly not applicable to minimal residual disease (MRD) stages. A flow cytometry‐based imaging approach was developed to detect chromosomal abnormalities following FISH in suspension (FISH‐IS), which enables the automated analysis of several log‐magnitude higher number of cells compared with the microscopy‐based approaches. This study demonstrates the applicability of FISH‐IS for detecting numerical chromosome aberrations, establishes accuracy, and sensitivity of detection compared with conventional FISH, and feasibility to study procured clinical samples of acute myeloid leukemia (AML). Male and female healthy donor peripheral blood mononuclear cells hybridized with combinations of chromosome enumeration probes (CEP) 8, X, and Y served as models for disomy, monosomy, and trisomy. The sensitivity of detection of monosomies and trisomies amongst 20,000 analyzed cells was determined to be 1% with a high level of precision. A high correlation (R2 = 0.99) with conventional FISH analysis was found based on the parallel analysis of diagnostic samples procured from 10 AML patients with trisomy 8 (+8). Additionally, FISH‐IS analysis of samples procured at the time of clinical remission demonstrated the presence of residual +8 cells indicating that this approach may be used to detect MRD and associated chromosomal defects. © 2012 International Society for Advancement of Cytometry

We investigated the ability of a plasmid-derived IL-21 delivered alone or in combination with the IL-15 gene to regulate immune responses to the HIV-1 envelope (Env) glycoprotein induced by DNA vaccination. Mice were injected with the gp140ΔCFIHXB2/89.6 vector expressing a modified Env glycoprotein with C-terminal mutations intended to mimic a fusion intermediate, in which the most divergent region encoding the variable V1, V2, and V3 domains of CXCR4-tropic HxB2 virus was replaced with the dual-tropic 89.6 viral strain. Using a recombinant vaccinia virus expressing 89.6 Env glycoprotein (vBD3) in a mouse challenge model, we observed that IL-21 plasmid produced sustained resistance to viral transmission when injected 5 days after DNA vaccination. Moreover, IL-21 in a synergistic manner with IL-15 expression vector augmented the vaccine-induced recall responses to the vBD3 challenge compared with those elicited by immunization in the presence of either cytokine alone. The synergistic combination of IL-21 and IL-15 plasmids promoted expansion of CD8+CD127+ memory T cell pools specific for a subdominant HLA-A2-restricted Env121–129 epitope (KLTPLCVTL). Our results also show that coimmunization with IL-21 and IL-15 plasmid combination resulted in enhanced CD8+ T cell function that was partially independent of CD4+ T cell help in mediating protection against vBD3 challenge. Furthermore, the use of IL-21 and IL-15 genes was able to increase Ab-dependent cellular cytotoxicity and complement-dependent lysis of Env-expressing target cells through augmentation of Env-specific IgG Ab levels. These data indicate that the plasmid-delivered IL-21 and IL-15 can increase the magnitude of the response to DNA vaccines.

The GD2 ganglioside expressed on neuroectodermal tumor cells is weakly immunogenic in tumor-bearing patients and induces predominantly IgM antibody responses in the immunized host. Using a syngeneic mouse challenge model with GD2-expressing NXS2 neuroblastoma, we investigated novel strategies for augmenting the effector function of GD2-specific antibody responses induced by a mimotope vaccine. We demonstrated that immunization of A/J mice with DNA vaccine expressing the 47-LDA mimotope of GD2 in combination with IL-15 and IL-21 genes enhanced the induction of GD2 cross-reactive IgG2 antibody responses that exhibited cytolytic activity against NXS2 cells. The combined immunization regimen delivered 1 day after tumor challenge inhibited subcutaneous (s.c.) growth of NXS2 neuroblastoma in A/J mice. The vaccine efficacy was reduced after depletion of NK cells as well as CD4(+) and CD8(+) T lymphocytes suggesting involvement of innate and adaptive immune responses in mediating the antitumor activity in vivo. CD8(+) T cells isolated from the immunized and cured mice were cytotoxic against syngeneic neuroblastoma cells but not against allogeneic EL4 lymphoma, and exhibited antitumor activity after adoptive transfer in NXS2-challenged mice. We also demonstrated that coimmunization of NXS2-challenged mice with the IL-15 and IL-21 gene combination resulted in enhanced CD8(+) T cell function that was partially independent of CD4(+) T cell help in inhibiting tumor growth. This study is the first demonstration that the mimotope vaccine of a weakly immunogenic carbohydrate antigen in combination with plasmid-derived IL-15 and IL-21 cytokines induces both innate and adaptive arms of the immune system leading to the generation of effective protection against neuroblastoma challenge.