Zinc Carnosine Metal-Organic Coordination Polymer as a Potent Broadly Active Influenza Vaccine Platform with In Vitro Shelf-Stability

Abstract

Current seasonal influenza vaccines are limited in that they need to be reformulated every year in order to account for the constant mutation of the virus. Hemagglutinin (HA) immunogens have been developed using a computationally optimized broadly reactive antigen (COBRA) methodology, which are able to elicit an antibody response that neutralizes antigenically distinct influenza strains; however, subunit proteins are not immunogenic enough on their own to generate a substantial immune response. Due to this, different delivery strategies and adjuvants can be used to improve immunogenicity. Recently, we reported a new coordination polymer composed of the dipeptide carnosine and zinc (ZnCar) that is able to deliver protein antigens along with CpG to generate a potent immune response. In the present work, ZnCar was used to deliver the COBRA HA immunogen Y2 and the adjuvant CpG. We incorporated Y2 into ZnCar using two different methods to assess which would be the most immunogenic. Mice vaccinated with Y2 and CpG complexed with ZnCar showed an improved humoral and cellular response when compared to mice vaccinated with soluble Y2 and CpG. Further, we demonstrate in vitro that when Y2 and CpG are coordinated with ZnCar, they are protected from degradation at 40 °C for 3 months or 24 °C for 6 months. Overall, ZnCar shows promise as a delivery vehicle for subunit vaccines, given its superior immunogenicity and in vitro storage stability.

Keywords: broadly active antigen; coordination polymer; influenza; storage stability; vaccine.

Figure 1.

Mice (n = 18) (BALB/cJ) were vaccinated on a prime + boost schedule (days 0 and 21) (IM) with the following: sucrose control, Y2 + CpG, Y2 + Addavax, Y2/ZnCar + ZnCar-CpG, or ZnCar-Y2 + ZnCar-CpG. Y2 and CpG were dosed at 1 and 10 μg, respectively. On days 14, 28, and 42 Y2- specific (A) IgG, (B) IgG1, and (C) IgG2a titers were assessed. (D) The ADCD activity of the sera was also analyzed for day 42. (E) The HAI titer was determined against four H1N1 viruses. (A-D) Data is represented as median ± range. (E) Data is represented as mean ± SD. ** = p ≤ 0.01, *** = p ≤ 0.001, and **** = p ≤ 0.0001.

Figure 2.

On day 35, mice (n = 5) were humanely euthanized to collect spleens and draining lymph nodes. Splenocytes were incubated with Y2 for 36 h after which the supernatant was collected to measure antigen specific (A) IL-2 and (B) IFN-γ secretion. (C) Lymph nodes were processed and stained with fluorescent antibodies to quantify GC B-cells (B220+, GL7+, and CD38-) via flow cytometry. (A-B) Data is represented as mean ± SD. (C) Data is represented as median ± range. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, and **** = p ≤0.0001.

Figure 3.

On day 56, mice (n = 13) were challenged with 100k pfu/mouse of A/California/07/2009 (H1N1). (A-B) Mice were weighed daily and mice that lost more than 20% of their initial body weight were humanely euthanized. (C) On day 59, mice (n = 3) were humanely euthanized, and their lung viral titers were assessed via a plaque assay. (A) # denotes significance between ZnCar-Y2 + ZnCar-CpG and Y2/ZnCar + ZnCar-CpG on day 5. (A) x denotes significance between Y2 + Addavax and Y2/ZnCar + ZnCar-CpG on day 5. (A) * denotes significance between ZnCar-Y2 + ZnCar-CpG and Y2 + CpG on day 5. Data is represented as mean ± SD. * = p ≤ 0.05, **** = p ≤0.0001, xxxx = p ≤0.0001, and #### = p ≤0.0001.

Figure 4.

SEM micrograph of (A-C) ZnCar-CpG, (D-F) ZnCar-Y2, and (G-I) Y2/ZnCar before storage or stored at either 40°C for 3 months or 24°C for 6 months. Scale bar represents 2 μm.

Figure 5.

ZnCar-CpG stored at various conditions along with CpG and ZnCar stored at −20°C were used to stimulate DC2.4 cells to determine whether the in vitro activity of CpG was maintained. After 24 h, the (A-B) percent viability and (C-D) TNF-α production were assessed. (A-B) Percent viability is measured by normalizing the experimental samples to an untreated control. (C) Statistical significance shown is for the comparison of ZnCar-CpG @ −20°C and ZnCar-CpG @ 40°C. (D) Statistical significance shown is for the comparison of ZnCar-CpG @ −20°C and ZnCar-CpG @ 24°C. Data is represented as mean ± SD. ns = p > 0.05, ** = p ≤ 0.01, and **** = p ≤0.0001.

Figure 6.

To assess the in vitro stability of Y2 in either ZnCar-Y2 or Y2/ZnCar stored at −20°C or 40°C for 3 months, a competitive ELISA was employed where the ability of the formulated Y2 to outcompete native Y2 for the binding to a Y2 specific antibody was measured. A library of four distinct Y2 specific antibodies consisting of (A) CA09–40, (B) CA09–28, (C) CA09–16, and (D) P1–05 was used to ensure that the structural integrity of Y2 after storage was maintained in diverse epitopes. Data is reported as the ELISA signal normalized to native Y2 as 100%. Data is represented as mean ± SD. ns = p > 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, and **** = p ≤0.0001.

Figure 7.

To assess the in vitro stability of Y2 in either ZnCar-Y2 or Y2/ZnCar stored at −20°C or 24°C for 6 months, a competitive ELISA was employed where the ability of the formulated Y2 to outcompete with native Y2 for the binding to a Y2 specific antibody was measured. A library of four distinct Y2 specific antibodies consisting of (A) CA09–40, (B) CA09–28, (C) CA09–16, and (D) P1–05 was used to ensure that the structural integrity of Y2 after storage was maintained in diverse epitopes. Data is represented as mean ± SD. ns = p > 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, and **** = p ≤0.0001.