Tumor stromal alternatively activated macrophages are important determinants of anti-tumor T lymphocyte responses, intratumoral neovascularization and metastatic dissemination. Our recent efforts to investigate the mechanism of macrophage migration inhibitory factor (MIF) in antagonizing anti-melanoma immune responses reveal that macrophage-derived MIF participates in macrophage alternative activation in melanoma-bearing mice. Both peripheral and tumor-associated macrophages (TAMs) isolated from melanoma bearing MIF-deficient mice display elevated pro-inflammatory cytokine expression and reduced anti-inflammatory, immunosuppressive and pro-angiogenic gene products compared to macrophages from tumor bearing MIF wildtype mice. Moreover, TAMs and myeloid-derived suppressor cells (MDSCs) from MIF-deficient mice exhibit reduced T lymphocyte immunosuppressive activities than do those from their wildtype littermates. Corresponding with reduced tumor immunosuppression and neoangiogenic potential by TAMs, MIF-deficiency confers protection against transplantable subcutaneous melanoma outgrowth and melanoma lung metastatic colonization. Finally, we report for the first time that our previously discovered MIF small molecule antagonist, 4-iodo-6-phenylpyrimidine (4-IPP), recapitulates MIF-deficiency in vitro and in vivo and attenuates tumor polarized macrophage alternative activation, immunosuppression, neoangiogenesis and melanoma tumor outgrowth. These studies describe an important functional contribution by MIF to tumor-associated macrophage alternative activation and provide justification for immunotherapeutic targeting of MIF in melanoma patients.

Display of peptides or proteins in an ordered, repetitive array, such as on the surface of a virus-like particle, is known to induce an enhanced immune response relative to vaccination with the "free" protein antigen. The coat protein of Tobacco mosaic virus (TMV) can accommodate short peptide insertions into the primary sequence, but the display of larger protein moieties as genetic fusions to the capsid protein has not been possible. We employed a randomized library approach to introduce a reactive lysine at the externally located amino terminus of the coat protein, which facilitated biotinylation of the capsid. To characterize display of heterologous proteins on the virion surface, we bound a model antigen (green fluorescent protein (GFP)-streptavidin (SA), expressed and purified from plants) to the biotinylated TMV particles, creating a GFP-SA decorated virus particle. A GFP-SA tetramer loading of 26% was obtained, corresponding to approximately 2200 GFP moieties displayed per intact virion. We evaluated the immunogenicity of GFP decorated virions in both mice and guinea pigs and found augmented humoral IgG titers in both species, relative to unbound GFP-SA tetramer. Next, we fused an N-terminal fragment of the Canine oral papillomavirus L2 protein to streptavidin. With TMV display, the L2 protein fragment was significantly more immunogenic than uncoupled antigen when tested in mice. By demonstrating the presentation of whole proteins, this study expands the utility of TMV as a vaccine scaffold beyond that which is possible by genetic manipulation.

h Griffithsin (GRFT) is a red-alga-derived lectin that binds the terminal mannose residues of N-linked glycans found on the surface of human immunodeficiency virus type 1 (HIV-1), HIV-2, and other enveloped viruses, including hepatitis C virus (HCV), severe acute respiratory syndrome coronavirus (SARS-CoV), and Ebola virus. GRFT displays no human T-cell mitogenic activity and does not induce production of proinflammatory cytokines in treated human cell lines. However, despite the growing evidence showing the broad-spectrum nanomolar or better antiviral activity of GRFT, no study has reported a comprehensive assessment of GRFT safety as a potential systemic antiviral treatment. The results presented in this work show that minimal toxicity was induced by a range of single and repeated daily subcutaneous doses of GRFT in two rodent species, although we noted treatment-associated increases in spleen and liver mass suggestive of an antidrug immune response. The drug is systemically distributed, accumulating to high levels in the serum and plasma after subcutaneous delivery. Further, we showed that serum from GRFT-treated animals retained antiviral activity against HIV-1-enveloped pseudoviruses in a cell-based neutralization assay. Overall, our data presented here show that GRFT accumulates to relevant therapeutic concentrations which are tolerated with minimal toxicity. These studies support further development of GRFT as a systemic antiviral therapeutic agent against enveloped viruses, although deimmunizing the molecule may be necessary if it is to be used in long-term treatment of chronic viral infections.

Griffithsin (GRFT), a lectin from Griffithsia species, inhibits human immunodeficiency virus-1 (HIV-1) replication at sub-nanomolar concentrations, with limited cellular toxicity. However, in vivo safety of GRFT is not fully understood, especially following parenteral administration. We first assessed GRFT’s effects in vitro, on mouse peripheral blood mononuclear cell (mPBMC) viability, mitogenicity, and activation using flow-cytometry, as well as cytokine secretion through enzyme-linked immunosorbent assay (ELISA). Toxicological properties of GRFT were determined after a single subcutaneous administration of 50 mg/kg or 14 daily doses of 10 mg/kg in BALB/c mice. In the context of microbicide development, toxicity of GRFT at 2 mg/kg was determined after subcutaneous, intravaginal, and intraperitoneal administrations, respectively. Interestingly, GRFT caused no significant cell death, mitogenicity, activation, or cytokine release in mPBMCs, validating the usefulness of a mouse model. An excellent safety profile for GRFT was obtained in vivo: no overt changes were observed in animal fitness, blood chemistry or CBC parameters. Following GRFT treatment, reversible splenomegaly was observed with activation of certain spleen B and T cells. However, spleen tissues were not pathologically altered by GRFT (either with a single high dose or chronic doses). Finally, no detectable toxicity was found after mucosal or systemic treatment with 2 mg/kg GRFT, which should be further developed as a microbicide for HIV prevention.

To assess the influence of mannosylated glycans on the immunogenicity of human immunodeficiency virus type 1 (HIV-1) Env proteins, we immunized mice with monomeric gp120 in the presence and absence of the mannosebinding protein, griffithsin (GRFT). For comparison, other groups of mice received the nonglycosylated HIV-1 Gag protein, with and without GRFT. Coimmunization with GRFT increased the anti-gp120 IgG reactivity significantly, but had no effect on the anti-Gag response. We also investigated the IgG response to GRFT and found that gp120, but not Gag, enhanced its immunogenicity. For both proteins, IgG1 antibodies dominated the IgG response, with IgG2b as the next most prevalent subclass. We conclude that gp120-GRFT complexes are more immunogenic than the free proteins, for both components, and that occluding the mannose moieties on monomeric gp120 can improve the humoral immune response to this protein.