Efficacious Recombinant Influenza Vaccines Produced by High Yield Bacterial Expression: A Solution to Global Pandemic and Seasonal Needs

It is known that physical linkage of TLR ligands and vaccine antigens significantly enhances the immunopotency of the linked antigens. We have used this approach to generate novel influenza vaccines that fuse the globular head domain of the protective hemagglutinin (HA) antigen with the potent TLR5 ligand, flagellin. These fusion proteins are efficiently expressed in standard E. coli fermentation systems and the HA moiety can be faithfully refolded to take on the native conformation of the globular head. In mouse models of influenza infection, the vaccines elicit robust antibody responses that mitigate disease and protect mice from lethal challenge. These immunologically potent vaccines can be efficiently manufactured to support pandemic response, pre-pandemic and seasonal vaccines

A West Nile virus recombinant protein vaccine that coactivates innate and adaptive immunity.

A chimeric protein West Nile virus (WNV) vaccine capable of delivering both innate and adaptive immune signals was designed by fusing a modified version of bacterial flagellin (STF2 Delta ) to the EIII domain of the WNV envelope protein. This fusion protein stimulated interleukin-8 production in a Toll-like receptor (TLR)-5-dependent fashion, confirming appropriate in vitro TLR5 bioactivity, and also retained critical WNV-E-specific conformation-dependent neutralizing epitopes as measured by enzyme-linked immunosorbent assay. When administered without adjuvant to C3H/HeN mice, the fusion protein elicited a strong WNV-E-specific immunoglobulin G antibody response that neutralized viral infectivity and conferred protection against a lethal WNV challenge. This potent EIII-specific immune response requires a direct linkage of EIII to STF2 Delta , given that a simple mixture of the 2 components failed to induce an antibody response or to provide protection against virus challenge. The presence of a functional TLR5 gene in vivo is also required--TLR5-deficient mice elicited only a minimal antigen-specific response. These results confirm that vaccines designed to coordinately regulate the innate and adaptive immune responses can induce protective immune responses without the need for potentially toxic adjuvants. They also support the further development of an effective WNV vaccine and novel monovalent and multivalent vaccines for related flaviviruses

Supplementary Figure 7 from Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory

A. Mean tumor burden over time of nude mice bearing OCSC1-F2 human ovarian tumors. mCherry fluorescent ROI was used to provide a measure of tumor burden. Data are presented as mean ± SEM. Treatments began on day 6 and continued through to day 12. B. Survival analysis using Kaplan-Meier.  </p

Supplementary Table S2 from Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory

Primers used for qPCR reactions</p

Supplementary Figure 4 from Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory

A. TKO mouse ovarian cancer cells were treated with increasing concentrations of CARG-2020 or VLV-GFP and effect on cell growth and cell death is quantified in real time using Cytation5/Biospa by measuring mCherry confluence and CelltoxTM fluorescence; B. Representative fluorescence images showing PBS Control and CARG-2020-treated cultures. red, mCherry; green, CelltoxTM; C. Human ovarian cancer cells, clone OCSC1-F2 and the hTERT-immortalized human endometrial stromal cells were treated with increasing concentrations of CARG-2020 for 72h and cell death was quantified using CelltoxTM fluorescence. Note that CARG-2020 induces cell death only in cancer cells and not in normal cells.</p

Supplementary Figure 3 from Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory

A. Diagram of CARG-2020 vector and GFP control vector; B. TKO mouse ovarian cancer cells were treated in vitro with PBS Control or 1x105 PFU/cell of CARG-2020 for 24h. IL12 mRNA was quantified by qPCR and secreted IL-12p40 and p70 protein were quantified using xMAP technology; C. Expression of IL-12 and IL-17R ECD were detected by western blot analysis in total cell lysate. D. Secretion of IL-17 RA-ECD was detected by western blot analysis in the supernatant of cells treated with CARG-2020. Control cells were treated with VLV-GFP vector. Representative images of 3 independent experiments. E. Expression of PDL-1 following treatment with CARG-2020. Note the time dependent decrease on PDL-1 expression on cells treated with CARG-2020. Control=VLV-GFP. Representative imagesof 3 independent experiments.</p

Supplementary Figure 2 from Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory

Diagram showing major immune phenotypes in each phase of ovarian tumor progression.</p

Supplementary Figure 1 from Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory

Flow cytometry gating strategy CD8 T cells, Macrophages and MDSC.</p

Supplementary Table S1 from Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory

Primers used to clone CARG-2020, VLV-12 and VLV-GFP</p