VaxCelerate II: rapid development of a self-assembling vaccine for Lassa fever

Abstract

Development of effective vaccines against emerging infectious diseases (EID) can take as much or more than a decade to progress from pathogen isolation/identification to clinical approval. As a result, conventional approaches fail to produce field-ready vaccines before the EID has spread extensively. Lassa is a prototypical emerging infectious disease endemic to West Africa for which no successful vaccine is available. We established the VaxCelerate Consortium to address the need for more rapid vaccine development by creating a platform capable of generating and pre-clinically testing a new vaccine against specific pathogen targets in less than 120 d A self-assembling vaccine is at the core of the approach. It consists of a fusion protein composed of the immunostimulatory Mycobacterium tuberculosis heat shock protein 70 (MtbHSP70) and the biotin binding protein, avidin. Mixing the resulting protein (MAV) with biotinylated pathogen-specific immunogenic peptides yields a self-assembled vaccine (SAV). To meet the time constraint imposed on this project, we used a distributed R&D model involving experts in the fields of protein engineering and production, bioinformatics, peptide synthesis/design and GMP/GLP manufacturing and testing standards. SAV immunogenicity was first tested using H1N1 influenza specific peptides and the entire VaxCelerate process was then tested in a mock live-fire exercise targeting Lassa fever virus. We demonstrated that the Lassa fever vaccine induced significantly increased class II peptide specific interferon-γ CD4(+) T cell responses in HLA-DR3 transgenic mice compared to peptide or MAV alone controls. We thereby demonstrated that our SAV in combination with a distributed development model may facilitate accelerated regulatory review by using an identical design for each vaccine and by applying safety and efficacy assessment tools that are more relevant to human vaccine responses than current animal models.

Keywords: 6MDP, 6-muramyl dipeptide; CGE, Capillary Gel Electrophoresis; CLO97, TLR7 ligand; CTL, Cytotoxic T-lymphocyte; CpG1826, Synthetic Oligodeoxynucleotide containing unmethylated dinucleotide sequences (Toll-like receptor 9 agonist); DARPA, Defense Advanced Research Projects Agency; EIDs, Emerging Infectious Diseases; Flu vaccine; GLP, Good Laboratory Practice; GMP, Good Manufacturing Practice; GP1, Glycoprotein-1; GP2, Glycoprotein-2; HLA, Human Leukocyte Antigen; HRP, Horseradish Peroxidase; LV, Lassa Fever Virus; Lassa fever virus; MAV, Mycobacterium tuberculosis Heat Shock Protein 70 – Avidin; MtbHSP70, Mycobacterium tuberculosis Heat Shock Protein 70; NHP, Non-human Primates; OVA, Ovalbumin; PAGE, Polyacrylamide Gel Electrophoresis; PBMC, Peripheral Blood Mononuclear Cell; PEG, Polyethyleneglycol; RVKR, Furin Cleavage Site (Arginine, Valine, Lysine, Arginine); SAV, Self-assembled vaccine; SAVL; Self-assembled vaccine formulated for Lassa Fever Virus; VaxCelerate; arenavirus; emerging infectious diseases; mycobacterium tuberculosis heat shock protein 70; peptide design; self-assembled vaccine; vaccine.

Figure 1.

Depiction of self-assembled vaccines structures. (A) Structure of MtbHSP70-avidin (MAV) and biotinylated antigens. (B) Structures of OVA peptides for SAV-OVA studies described in Table 2A. (C) and (D) Structures of short (C) and long (D) Flu peptides described in Table 2B for SAV1 and SAV2 used in Flu study.

Figure 2.

Characterization of Pfēnex’ MtbHSP70-avidin (MAV). (A) 500 nanograms of MAV were electrophoresed on a 4–12% Bis-Tris NuPAGE gel run in MOPS buffer at constant 200 V for 1 hr. (B) Reproduction of the capillary gel electrophoresis (CGE) profile provided by Pfēnex. 150 nl of purified MAV (976.2 ng/nl) were injected into the microfluidic channel of the LabChip GX/II using electrokinetic injection. Applying a voltage across the channel effected protein separation. Protein bands were detected via laser induced fluorescence. Arrows indicate the migration position of MtbHSP70-avidin. (C) MtbHSP70-avidin ATPase activity assessment. A series of MtbHSP70-avidin concentrations (25 – 500 ng) were incubated as described in materials and methods. (D, E, F and G) Flow cytometric analysis of splenocytes and lymph node cells from ovalbumin immunized mice as described in Materials and Methods. Splenocytes (D, F) and lymph node cells (E, G) were prepared as described in Materials and Methods and stimulated with medium alone, SIINFEKL (D, E; 10 μg/ml) or ISQAVHAAHAEINEAGR (F, G; 10 μg/ml) for 24 hours at 37°C. The percent of CD3⁺CD8⁺IFN-γ⁺ and CD3⁺CD4⁺IFN-γ⁺ cells above medium alone was determined and plotted as percent above control. (H) Gel-shift assay of SAV1 and SAV2. 400 to 800 ng of protein were denatured and subjected to electrophoresis on a Novex® 4–12% Bis-Tris NuPAGE gel run in MOPS buffer at constant 200 V for 5 hr. Arrows indicate MAV, SAV1 and SAV2. (I) SAV1 and SAV2 ATPase activity. Three different concentrations of MAV, SAV1 and SAV2 were assayed for their ability to hydrolyze ATP as described in Materials and Methods. Abbreviations: MAV - Mycobacterium tuberculosis heat shock protein 70 fused to avidin; OVA - ovalbumin; PEG - polyethylene glycol; SAV1 - MAV assembled with flu specific peptides 1–4 (Table 2B); SAV2 - MAV assembled with flu specific peptides 5 and 6 (Table 2B).

Figure 3.

Flow cytometric analysis of splenocytes and lymphocytes from FluVax, SAV1 and SAV2 immunized mice. Splenocytes and lymphocytes were prepared as described in Materials and Methods and stimulated with the indicated flu peptides (10 μg/ml) or medium alone for 24 hours at 37°C. Brefeldin A was added 4 hours prior to harvest to inhibit protein transport. Cells were stained for CD3, CD4, CD8, IL-4 and interferon-γ fixed and analyzed on a BD LSR Fortessa^TM. The percent of CD3⁺CD4⁺IFN-γ⁺ cells above medium alone was determined and plotted as percent above control. (A) Splenocytes response, (B) lymph node cells response. The percent of CD3⁺CD4⁺IL-4⁺ cells above medium alone was determined and plotted as percent above control for (C) splenocytes response, (D) lymph node cells response. Numbers in parenthesis indicate non-responders.

Figure 4.

Structure and sequences of Lassa fever virus immunogenic peptides. (A) Basic framework for the synthesis of immunogenic concatenated class I and class II peptides. (B) Biotinylated Lassa fever virus specific peptides.

Figure 5.

Characterization of the self-assembled Lassa fever virus vaccine (SAVL). A) ATPase activity of MAV, MAVf, and SAVL using 25, 50 and 100 ng. The assay was carried out as described in Materials and Methods. (B) Flow cytometric analysis of splenocytes from mice immunized with SAVL. Splenocytes from mice immunized with PBS, peptides, MAVf and SAVL were prepared as described and stimulated with medium alone or a pool of class II peptides (10 μg/ml). Total incubation time was 24 hours at 37°C. Brefeldin A was added 4 hours prior to harvest. (C) Comparison of splenocytes response to class II Lassa fever virus specific peptides between different immunization groups.

Figure 6.

VaxCelerate time line.