Enhancement of Mucosal Immunization with Virus-Like Particles of Simian Immunodeficiency Virus

Abstract

Cholera toxin (CT) is the most potent known mucosal adjuvant, but its toxicity precludes its use in humans. Here, in an attempt to develop safe and effective mucosal adjuvants, we compared immune responses to simian immunodeficiency virus (SIV) virus-like particles (VLPs) after intranasal coimmunization with RANTES, CpG oligodeoxynucleotides (ODN), or CT. Antibody analysis demonstrated that RANTES and CpG ODN had capacities for mucosal adjuvanticity, i.e., for enhancing serum and vaginal antibodies specific to SIV Env, similar to those for CT. RANTES and CpG ODN skewed serum antibodies predominantly to the immunoglobulin G2a isotype. Most importantly, RANTES and CpG ODN were more effective than CT in increasing neutralizing titers of both serum and vaginal antibodies. After intranasal coadministration with VLPs, RANTES or CpG ODN also induced increased levels of gamma interferon (IFN-␥)-producing lymphocyte and cytotoxic T-lymphocyte activities in both spleen and lymph nodes but did not increase the levels of interleukin-4-producing lymphocytes. The results suggest that RANTES and CpG ODN enhance immune responses in a T-helper-cell-type-1 (Th1)-oriented manner and that they can be used as effective mucosal adjuvants for enhancing both humoral and cellular immune responses in the context of VLPs, which are particulate antigens.

Incorporation of Glycosylphosphatidylinositol-Anchored Granulocyte- MacrophageColony-Stimulating Factor or CD40 Ligand Enhances Immunogenicity of Chimeric Simian Immunodeficiency Virus-Like Particles

“…We previously produced SIV VLPs by expression of the Gag and Env proteins in insect cells using rBVs as expression vectors (22,57). Here, we designed membrane-anchored forms of GM-CSF to enable its incorporation into VLPs containing SIV Env and Gag as an approach to induce enhanced immune responses against SIV antigens.…”

“…All sera were individually collected, and SIV Env-specific-antibody levels for IgG, IgG1, IgG2a, IgG2b, and IgG3 were quantitatively determined by ELISAs as previously described (22). The substrate OPD (Zymed, San Francisco, CA) dissolved in citrate buffer, pH 5.0, was used to develop color.…”

“…Cytokine levels were determined according to the manufacturer's instructions. For ELISPOT assay, freshly isolated splenocytes (0.5 to 1.0 ϫ 10 6 /200 l complete RPMI) from immunized mice were cultured for 36 h in the presence of peptide stimulants in complete RPMI medium, as previously described (22). All ELISPOT reagents were purchased from BD-PharMingen.…”

“…Optical density was read at 450 nm, and antibody concentrations were determined based on standard curves of each subtype antibodies. Neutralization activity was determined using SMAGI cell assays as described previously (22). Briefly, preimmune and immune sera were heat inactivated at 56°C for 30 min, serially diluted, incubated with SIVmac1A11 virus (100 PFU) for 1 h at 37°C, and then added to the SMAGI cells.…”

“…Spleens were collected from individual mice at 2 weeks after the final immunization, and a single-cell suspension was prepared and used for ELISPOT assays and cytokine ELISAs as described previously (22). Spleen cells or mesenteric lymph node cells (0.2 ϫ 10 6 /200 l complete RPMI medium) were prepared from immunized mice at 2 weeks after the last immunization and stimulated in vitro with Env peptide pools at a final concentration of 1 g/ml in complete RPMI medium.…”