Red-Shifted Aequorin Variants Incorporating Non-Canonical Amino Acids: Applications in \u3cem\u3eIn Vivo\u3c/em\u3e Imaging

The increased importance of in vivo diagnostics has posed new demands for imaging technologies. In that regard, there is a need for imaging molecules capable of expanding the applications of current state-of-the-art imaging in vivo diagnostics. To that end, there is a desire for new reporter molecules capable of providing strong signals, are non-toxic, and can be tailored to diagnose or monitor the progression of a number of diseases. Aequorin is a non-toxic photoprotein that can be used as a sensitive marker for bioluminescence in vivo imaging. The sensitivity of aequorin is due to the fact that bioluminescence is a rare phenomenon in nature and, therefore, it does not suffer from autofluorescence, which contributes to background emission. Emission of bioluminescence in the blue-region of the spectrum by aequorin only occurs when calcium, and its luciferin coelenterazine, are bound to the protein and trigger a biochemical reaction that results in light generation. It is this reaction that endows aequorin with unique characteristics, making it ideally suited for a number of applications in bioanalysis and imaging. Herein we report the site-specific incorporation of non-canonical or non-natural amino acids and several coelenterazine analogues, resulting in a catalog of 72 cysteine-free, aequorin variants which expand the potential applications of these photoproteins by providing several red-shifted mutants better suited to use in vivo. In vivo studies in mouse models using the transparent tissue of the eye confirmed the activity of the aequorin variants incorporating L-4-iodophehylalanine and L-4-methoxyphenylalanine after injection into the eye and topical addition of coelenterazine. The signal also remained localized within the eye. This is the first time that aequorin variants incorporating non-canonical amino acids have shown to be active in vivo and useful as reporters in bioluminescence imaging

Therapy of established B16-F10 melanoma tumors by a single vaccination of CTL/T helper peptides in VacciMax(®)

BACKGROUND: Melanoma tumors are known to express antigens that usually induce weak immune responses of short duration. Expression of both tumor-associated antigens p53 and TRP2 by melanoma cells raises the possibility of simultaneously targeting more than one antigen in a therapeutic vaccine. In this report, we show that VacciMax(® )(VM), a novel liposome-based vaccine delivery platform, can increase the immunogenicity of melanoma associated antigens, resulting in tumor elimination. METHODS: C57BL/6 mice bearing B16-F10 melanoma tumors were vaccinated subcutaneously 6 days post tumor implantation with a mixture of synthetic peptides (modified p53: 232–240, TRP-2: 181–188 and PADRE) and CpG. Tumor growth was monitored and antigen-specific splenocyte responses were assayed by ELISPOT. RESULTS: Vaccine formulated in VM increased the number of both TRP2- and p53-specific IFN-γ producing splenocytes following a single vaccination. Vaccine formulated without VM resulted only in enhanced IFN-γ producing splenocytes to one CTL epitopes (TRP2:180–188), suggesting that VM overcomes antigen dominance and enhances immunogenicity of multiple epitopes. Vaccination of mice bearing 6-day old B16-F10 tumors with both TRP2 and p53-peptides formulated in VM successfully eradicated tumors in all mice. A control vaccine which contained all ingredients except liposomes resulted in eradication of tumors in no more than 20% of mice. CONCLUSION: A single administration of VM is capable of inducing an effective CTL response to multiple tumor-associated antigens. The responses generated were able to reject 6-day old B16-F10 tumors

Rejection of large HPV-16 expressing tumors in aged mice by a single immunization of VacciMax® encapsulated CTL/T helper peptides

The incidence of cancer increases significantly in later life, yet few pre-clinical studies of cancer immunotherapy use mice of advanced age. A novel vaccine delivery platform (VacciMax®,VM) is described that encapsulates antigens and adjuvants in multilamellar liposomes in a water-in-oil emulsion. The therapeutic potential of VM-based vaccines administered as a single dose was tested in HLA-A2 transgenic mice of advanced age (48–58 weeks old) bearing large palpable TC1/A2 tumors. The VM-based vaccines contained one or more peptides having human CTL epitopes derived from HPV 16 E6 and E7. VM formulations contained a single peptide, a mixture of four peptides or the same four peptides linked together in a single long peptide. All VM formulations contained PADRE and CpG as adjuvants and ISA51 as the hydrophobic component of the water-in-oil emulsion. VM-formulated vaccines containing the four peptides as a mixture or linked together in one long peptide eradicated 19-day old established tumors within 21 days of immunization. Peptide-specific cytotoxic cellular responses were confirmed by ELISPOT and intracellular staining for IFN-γ producing CD8+ T cells. Mice rendered tumor-free by vaccination were re-challenged in the opposite flank with 10 million HLF-16 tumor cells, another HLA-A2/E6/E7 expressing tumor cell line. None of these mice developed tumors following the re-challenge. In summary, this report describes a VM-formulated therapeutic vaccine with the following unprecedented outcome: a) eradication of large tumors (> 700 mm3) b) in mice of advanced age c) in less than three weeks post-immunization d) following a single vaccination

A Targeted and Adjuvanted Nanocarrier Lowers the Effective Dose of Liposomal Amphotericin B and Enhances Adaptive Immunity in Murine Cutaneous Leishmaniasis

Background: Amphotericin B (AmB), the most effective drug against leishmaniasis, has serious toxicity. As Leishmania species are obligate intracellular parasites of antigen presenting cells (APC), an immunopotentiating APC-specific AmB nanocarrier would be ideally suited to reduce the drug dosage and regimen requirements in leishmaniasis treatment. Here, we report a nanocarrier that results in effective treatment shortening of cutaneous leishmaniasis in a mouse model, while also enhancing L. major specific T-cell immune responses in the infected host. Methods: We used a Pan-DR-binding epitope (PADRE)-derivatized-dendrimer (PDD), complexed with liposomal amphotericin B (LAmB) in an L. major mouse model and analyzed the therapeutic efficacy of low-dose PDD/ LAmB vs full dose LAmB. Results: PDD was shown to escort LAmB to APCs in vivo, enhanced the drug efficacy by 83% and drug APC targeting by 10-fold and significantly reduced parasite burden and toxicity. Fortuitously, the PDD immunopotentiating effect significantly enhanced parasite-specific T-cell responses in immunocompetent infected mice. Conclusions: PDD reduced the effective dose and toxicity of LAmB and resulted in elicitation of strong parasite specific T-cell responses. A reduced effective therapeutic dose was achieved by selective LAmB delivery to APC, bypassing bystander cells, reducing toxicity and inducing antiparasite immunity

Studies on the roles of T helper type I and type II cytokines in HIV immunopathogenesis: Role and regulation of interleukin-10.

Infection of immune cells with human immunodeficiency virus (HIV) induces dysregulation of cytokines which may play a vital role in HIV pathogenesis. I analyzed the expression of Th1 (interferon-$\gamma,$ (IFN-$\gamma)\rbrack$ and Th2 (interleukin-4 (IL-4), IL-10) type cytokines in unstimulated and mitogen stimulated peripheral blood mononuclear cells (PBMC) from HIV seropositive (HIV\sp+) patients. It was determined that IFN-$\gamma$ mRNA in unstimulated PBMC was significantly decreased and IL-10 mRNA as well as IL-10 protein was significantly increased in patients with 400 CD4$\sp+$ T cells/mm$\sp3$ (n = 6) and normal controls (n = 16). Mitogen stimulation of PBMC revealed two groups of HIV$\sp+,$ low and normal IL-10 producers. Production of IL-4 was reduced in HIV$\sp+$ individuals with 400 CD4\sp+ T cells/mm\sp3. However, ability to produce IFN-y by mitogen stimulated PBMC and CD4 T\sp+ cells was not impaired in HIV\sp+ individuals. These results suggest that PBMC of HIV\sp+ exhibit dysregulation of Th2 type cytokines which may play a role in HIV immunopathogenesis. In the next set of experiments, the IL-10 production was correlated with the levels of proliferative responses to recall antigens. Low IL-10 producers proliferated in response to recall antigens, and demonstrated enhanced recall antigen-induced proliferation upon addition of anti-IL-10 antibodies and/or IL-12. Conversely, normal IL-10 producers had PBMC that failed to proliferate to recall antigens, and did not demonstrate enhanced recall antigen-induced proliferation upon addition of anti-IL-10 antibodies and/or IL-12. Source of the IL-10 production in PBMC of HIV\sp+ individuals was shown to be monocytes, while, in HIV controls, it was produced by both T cells and monocytes. The molecular mechanisms underlying the production of IL-10 are not clear. I have demonstrated that monocytes/macrophages are required for IL-10 production by normal activated T cells. IL-10 production was significantly downregulated in both T cell and monocyte depleted PBMC compared to undepleted PBMC, and IL-10 production could be restored following addition of monocyte conditioned medium (MCM), this suggested that IL-10 production by T cells is regulated by monokine(s) produced by activated monocytes. The monokine(s) responsible for IL-10 induction by T cells were further studied. Addition of IL-6 and IL-12 enhanced IL-10 production in monocyte depleted PBMC in a dose dependent and additive manner. With respect to regulation of IL-10 produced by monocytes, tumor necrosis factor $\alpha$ (TNF-$\alpha)$ was found to induce IL-10 production by resting purified monocytes. Taken together, these findings suggest that IL-10 production by human T cells and monocytes is differentially regulated. IL-12 and IL-6 induce the expression of IL-10 by PHA stimulated T cells whereas TNF-$\alpha$ induces IL-10 production by monocytes. Since IL-10 inhibits production of IL-6, IL-12 and TNF-$\alpha,$ these results may indicate a potential mechanism of negative feedback regulation of the immune system. Furthermore, mitogen stimulated PBMC from HIV\sp+ individuals produced significantly lower levels of IL-12 than did those from HIV-controls. A defect in IL-12 induction may partially cause IL-10 dysregulation in HIV infection

Non-small-cell lung cancer homing peptide-labeled dendrimers selectively transfect lung cancer cells

Lung cancer gene therapies require reagents to selectively transfect lung tumors after systemic administration. We created a reagent called NSCLC-NP by attaching a peptide with binding affinity for lung cancer to polyamidoamine dendrimers. The positively charged dendrimers electrostatically bind negatively charged nucleic acids, inhibit endogenous nucleases and transfect cells targeted by the attached peptide. In vitro, NSCLC-NP complexed to DNA plasmids bound and transfected three human lung cancer cell lines producing protein expression of the plasmid's gene. In vivo, systemically administered NSCLC-NP selectively transfected lung cancer cells growing in RAG1KO mice. The capability of NSCLC-NP to selectively transfect lung cancer allows its future use as a vehicle to implement human lung cancer gene therapy strategies

A Targeted and Adjuvanted Nanoparticle for Immunochemotherapy of Leishmania Infections

Targeted drug delivery is critical for minimizing off-target toxicity by chemotherapeutic drugs. Amphotericin-B is an effective anti-leishmaniasis agent that induces significant nephrotoxicity. Complexing amphotericin-B with liposomes (AmBisome, LAmB) reduces nephrotoxicity and improves LAmB efficacy in treating leishmaniasis in humans. However, complicated dosing regimens are required to minimize side effects. Given that Leishmania species are obligate parasites in phagocytes, selectively targeting LAmB to infected phagocytes would likely improve efficacy, decrease systemic toxicity, and simplify dosing regimens. To target LAmB to phagocytes, we developed a PADRE-derivatized-dendrimer (PDD)-LAmB nanocarrier platform by functionalizing dendrimers that complex LAmB with a peptide that targets major histocompatibility complex (MHC) class II receptors. MHC class II receptor expression increases on phagocytes during the course of leishmaniasis, further enhancing infected cells as targets for PDD-LAmB. In a murine model for Leishmania major infection, PDD-LAmB efficiently targeted infected phagocytic cells, reducing the LAmB effective dose by 80 %, with improved pharmacokinetics. Moreover, the PDD MHC class II-targeting peptide is a universal helper T-cell epitope, allowing PDD-LAmB complexes to elicit parasite-specific T-cell responses. Thus, PDD-LAmB complex is a promising candidate for further development and human clinical trials, reducing and simplifying LAmB dosing, reducing side effects, and stimulating T-cell responses

Gene-based vaccination and screening methods to develop monoclonal antibodies

Gene-based in vivo electroporation has the potential to be used as a "protein-free" method to elicit immune responses and to generate monoclonal antibodies (mAb) against proteins/peptides in hosts. However, the method is very useful to raise mAbs against proteins and peptides and not for carbohydrates, lipids, or haptens. Nevertheless, making mAb using this potent method faces a few challenges: the parameters of the electroporation needs further standardized, the final boost still needs protein antigens, and the primary screening of the clones requires purified protein. We present methods to overcome these challenges by an optimized electroporation framework and a method to use transiently transfected cells for the final boost, as well as for screening of the resulting clones via the use of an "In-Cell Western" method

APC-Targeted (DNA) Vaccine Delivery Platforms: Nanoparticle Aided

Only a small fraction of any administered drug, including a vaccine, reaches its intended target tissue or cells. Homing vaccines to antigen-presenting cells (APC), where they can do their magic, has been the focus of attention for several decades. Since they are equipped with proper co-stimulatory signals, only professional APC are able to correctly process antigens and stimulate T and B cells to mount specific immune responses. With advances in the fields of modern immunology and nanotechnology, the new vaccine delivery platforms are emerging. APC-targeted vaccine delivery is required to elicit protective immunity, to reduce manufacturing costs, to minimize unanticipated effects of vaccines caused by off-target effects (thus reducing immunosuppressive mechanisms and toxicity), and to dramatically increase immunization efficacy. Indeed, in vivo targeting of APCs may represent the best hope for the generation of strategies leading to a personalized immunization without the need of expensive and complex ex vivo manipulation of patients’ PBMCs. Here, we will review and compare novel approaches on nanoparticle-based-targeted vaccine platforms, evaluating their molecular mechanisms of action, their translation ability, their efficacy, and their cost