Chemical-free inactivated whole influenza virus vaccine prepared by ultrashort pulsed laser treatment

There is an urgent need for rapid methods to develop vaccines in response to emerging viral pathogens. Whole inactivated virus (WIV) vaccines represent an ideal strategy for this purpose; however, a universal method for producing safe and immunogenic inactivated vaccines is lacking. Conventional pathogen inactivation methods such as formalin, heat, ultraviolet light, and gamma rays cause structural alterations in vaccines that lead to reduced neutralizing antibody specificity, and in some cases, disastrous T helper type 2-mediated immune pathology. We have evaluated the potential of a visible ultrashort pulsed (USP) laser method to generate safe and immunogenic WIV vaccines without adjuvants. Specifically, we demonstrate that vaccination of mice with laser-inactivated H1N1 influenza virus at about a 10-fold lower dose than that required using conventional formalin-inactivated influenza vaccines results in protection against lethal H1N1 challenge in mice. The virus, inactivated by the USP laser irradiation, has been shown to retain its surface protein structure through hemagglutination assay. Unlike conventional inactivation methods, laser treatment did not generate carbonyl groups in protein, thereby reducing the risk of adverse vaccine-elicited T helper type 2 responses. Therefore, USP laser treatment is an attractive potential strategy to generate WIV vaccines with greater potency and safety than vaccines produced by current inactivation techniques

Cancer Immunotherapy Employing an Innovative Strategy to Enhance CD4+ T Cell Help in the Tumor Microenvironment

<div><p>Chemotherapy and/or radiation therapy are widely used as cancer treatments, but the antitumor effects they produce can be enhanced when combined with immunotherapies. Chemotherapy kills tumor cells, but it also releases tumor antigen and allows the cross-presentation of the tumor antigen to trigger antigen-specific cell-mediated immune responses. Promoting CD4+ T helper cell immune responses can be used to enhance the cross-presentation of the tumor antigen following chemotherapy. The pan HLA-DR binding epitope (PADRE peptide) is capable of generating antigen-specific CD4+ T cells that bind various MHC class II molecules with high affinity and has been widely used in conjunction with vaccines to improve their potency by enhancing CD4+ T cell responses. Here, we investigated whether intratumoral injection of PADRE and the adjuvant CpG into HPV16 E7-expressing TC-1 tumors following cisplatin chemotherapy could lead to potent antitumor effects and antigen-specific cell-mediated immune responses. We observed that treatment with all three agents produced the most potent antitumor effects compared to pairwise combinations. Moreover, treatment with cisplatin, CpG and PADRE was able to control tumors at a distant site, indicating that our approach is able to induce cross-presentation of the tumor antigen. Treatment with cisplatin, CpG and PADRE also enhanced the generation of PADRE-specific CD4+ T cells and E7-specific CD8+ T cells and decreased the number of MDSCs in tumor loci. The treatment regimen presented here represents a universal approach to cancer control.</p></div

Local immune response to PADRE peptide and tumor antigen (HPV16 E7).

<p>C57BL/6 mice (5 per group) were challenged subcutaneously with TC-1 tumor cells and treated with various combinations of cisplatin, CpG and PADRE peptide as indicated. 12 days after the last antigen delivery, tumor-infiltrating lymphocytes were harvested to analyze the immune cell subsets. A. Representative flow cytometry analysis depicting the frequency of PADRE-specific IFN-γ-secreting CD4+ T cells among the total tumor infiltrating CD4+ T cells. B. Bar graph quantification of data from A. (mean ± S.E.). C. Representative flow cytometry analysis depicting the absolute number of E7 tetramer-binding CD8+ T cells in 5×10⁵ single-cell suspension prepared from tumors. D. Bar graph quantification of data from C. (mean ± S.E.).</p

Local intratumoral treatment triggers systemic antitumor effects.

<p>C57BL/6 mice (5 mice/group) were challenged sequentially with TC-1 tumor cells on day 0 (right flank) and day 5 (left flank), and then were either untreated or treated with cisplatin combined with intratumoral injection with CpG and PADRE peptide in the primary tumor. In the CD8+ T cell depletion group, mice were treated as above, with the adition of 100 µg anti-mouse CD8 antibody (clone 2.43) injected intraperitoneally on days 3, 4 and 5 after tumor challenge, and then 150 µg per week subsequently. A. Schematic diagram of the treatment regimen. B. Scatter plot of primary tumor growth kinetics. C. Scatter plot of secondary tumor growth kinetics. D. Kaplan-Meier survival plot. (***<i>p</i><0.001, ****<i>p</i><0.0001).</p

Antitumor effect in TC-1 tumor-bearing mice treated with intratumoral injection of irrelevant peptide combined with chemotherapy and CpG.

<p>C57BL/6 mice (5 mice/group) were challenged subcutaneously with TC-1 tumor cells and treated with various combinations of cisplatin, CpG and PADRE peptide. A. Schematic diagram of the treatment regimens. B. Plot of tumor growth kinetics. C. Kaplan-Meier survival plot. (*<i>p</i><0.05, ***<i>p</i><0.001, ***<i>p</i><0.0001).</p

Activated CD4+ T cells directly induced apoptosis of MDSCs.

<p>Purified splenic CD11b+ Ly6G^Hi MDSCs were incubated with PADRE-specific CD4+ T cells or medium control for 24 hours. A. Representative flow cytometry plots depicting the frequency of CD11b+ Ly6G^Hi Annexin V+ MDSCs. B. Bar graph quantification of the data A (mean ± S.E.) (****<i>p</i><0.0001).</p

Treatment of Tumors with Vitamin E Suppresses Myeloid Derived Suppressor Cells and Enhances CD8+ T Cell-Mediated Antitumor Effects

<div><p>Vitamin E has been shown to have strong anticarcinogenic properties, including antioxidant characteristics, making it an ideal candidate for use in combination with immunotherapies that modify the tumor microenvironment. The tumor microenvironment contains immunosuppressive components, which can be diminished, and immunogenic components, which can be augmented by immunotherapies in order to generate a productive immune response. In the current study, we employ the α-tocopherol succinate isomer of vitamin E to reduce immunosuppression by myeloid derived suppressor cells (MDSCs) as well as adoptive transfer of antigen-specific CD8+ T cells to generate potent antitumor effects against the HPV16 E7-expressing TC-1 tumor model. We show that vitamin E alone induces necrosis of TC-1 cells and elicits antitumor effects in TC-1 tumor-bearing mice. We further demonstrate that vitamin E reverses the suppression of T cell activation by MDSCs and that this effect is mediated in part by a nitric oxide-dependent mechanism. Additionally, treatment with vitamin E reduces the percentage of MDSCs in tumor loci, and induces a higher percentage of T cells, following T cell adoptive transfer. Finally, we demonstrate that treatment with vitamin E followed by E7-specific T cell adoptive transfer experience elicits potent antitumor effects in tumor-bearing mice. Our data provide additional evidence that vitamin E has anticancer properties and that it has promise for use as an adjuvant in combination with a variety of cancer therapies.</p></div

Characterization of the T-cell accumulation in tumor-bearing mice treated with Vitamin E.

<p>1×10⁵ TC-1 cells were injected subcutaneously into wild type C57BL/6 mice. 10 days later, mice were treated as in (A) and one day after the last vitamin E treatment, luciferase-expressing E7-specific CD8+ T-cells were adoptively transferred using intravenous injection. (<b>A</b>) Schematic diagram of the treatment regimen. (<b>B</b>) Bar graph depicting the fluorescence intensity in tumor-bearing mice treated with vitamin E or DMSO (mean ± S.D.).</p