Aleksandra Kowalczyk

DNA Vaccine Expressing the Mimotope of GD2 Ganglioside Induces Protective GD2 Cross-reactive Antibody Responses

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 monoclonal antibody (mAb) 14G2a led to isolation of mimetic peptide 47, which inhibited the binding of 14G2a antibody to GD2-positive tumor cells. The peptide was also recognized by GD2-specific serum antibodies from a patient with neuroblastoma, suggesting that it bears an internal image of GD2 ganglioside expressed on the tumor cells. The molecular basis for antigenicity of the GD2 mimetic peptide, established by molecular modeling and mutagenesis studies, led to the generation of a 47-LDA mutant with an increased mimicry to GD2. Immunization of mice with peptide 47-LDA-encoded plasmid DNA elicited GD2 cross-reactive IgG antibody responses, which were increased on subsequent boost with GD2 ganglioside. The vaccine-induced antibodies recognized GD2-positive tumor cells, mediated complement-dependent cytotoxicity, and exhibited protection against s.c. human GD2-positive melanoma growth in the severe combined immunodeficient mouse xenograft model. The results from our studies provide insights into approaches for boosting GD2 cross-reactive IgG antibody responses by minigene vaccination with a protective epitope of GD2 ganglioside.

Increased Level and Longevity of Protective Immune Responses Induced by DNA Vaccine Expressing the HIV-1 Env Glycoprotein when Combined with IL-21 and IL-15 Gene Delivery

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.

RNActive® Technology: Generation and Testing of Stable and Immunogenic mRNA Vaccines

Developing effective mRNA vaccines poses certain challenges concerning mRNA stability and ability to induce sufficient immune stimulation and requires a specific panel of techniques for production and testing. Here, we describe the production of stabilized mRNA with enhanced immunogenicity, generated using conventional nucleotides only, by introducing changes to the mRNA sequence and by complexation with the nucleotide-binding peptide protamine (RNActive® technology). Methods described here include the synthesis, purification, and protamine complexation of mRNA vaccines as well as a comprehensive panel of in vitro and in vivo methods for evaluation of vaccine quality and immunogenicity.