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

Figures & data

Table 1. Biotin binding assay of MAV and SAV

A. MtbHSP70avidin biotin binding assessment*
Condition	OD 450–650 nm
Biotin-HRP	0.33 +/− 0.12
Biotin-HRP + MtbHSP70avidin	2.35 +/− 0.24
B. Percent self-assembly of biotinylated peptides
Peptide #	OD 450 – background	% completion
1	0.01 +/− 0.014	99.0
3	0.012 +/− 0.003	98.8
4	0.015 +/− 0.001	98.5
5	0.005 +/− 0.007	99.9

*Biotin-HRP was incubated with or without MtbHSP70-avidin. Unbound biotin-HRP was removed by addition of magnetic Streptavidin beads. An aliquot of the resulting supernatant was mixed with Tetramethylbenzidine and incubated in the dark for 5 minutes. The reaction was stopped with 2N H₂SO₄ and reaction read at 450 nm after subtracting background at 650 nm. All reactions were performed in duplicates

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).

Table 2. Epitopes and peptides sequences used for OVA, Flu and Lassa fever virus vaccines

Table 3. Immunization schemes

Table 4. Antibody responses against immunizing components of the vaccines*

A) FluVax Pilot experiment: Median (min; max)
Antigens\Immunization group	MAV	FluVax	SAV1	SAV2
FluVax Peptides	ND**	5756	ND	ND
		(0, 9243)
MtbHSP70	55735 (50205; 61815)	ND	6214 (60739; 63466)	6311 (55941; 6352)
B) Lassa Fever vaccine study: Median (min; max)
Antigens\Immunization group	Saline	Peptides	MAVf	SAVL
Lassa Specific peptides	ND	1671***	ND	1753***
MtbHSP70	ND	ND	59622 (54908; 66679)	59559 (27842; 62856)

*Endpoint titers using the method of Frey, A, Di Canzio, J., & Zurakowski, D. (1998). Titers determine as highest sera dilutions crossing the 95% confidence interval cutoff curve.

**ND; none determine. Sera dilutions at 1:100 yielded results below the 95% confidence interval curve.

***Endpoint titer for pooled sera against peptide S6 (Fig. 4B). No titers were observed against the other peptides.

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.

Table 5. Comparison of T cell immune responses observed in this study with other EID vaccines

Pathogen	Vaccine Type	Type of Subject	Immuno-assay	Response	Citation
Lassa	Protein-peptide complex	Tg HLA-DR3 mice	Flow cytometry	891 cells per 10^6 splenocytes; 0.59% of total CD4^+ T cells	Current study
Dengue	Attenuated virus	Humans	Flow cytometry	Up to 0.16% of CD4^+ T cells	See ref. ^77
Hepatitis C	Adjuvanted proteins	BALB/c mice	ELISpot	125–200 cells per 10^6 splenocytes	See ref. ^78
HIV	BCG expression Gag	Chacma baboons	ELISpot	0.4% of CD4^+ T CELLS	See ref. ^79
Smallpox	Attenuated live virus	BALB B/c mice	Flow cytometry	0.1–0.3% of CD4^+ T cells	See ref. ^80
Yellow fever	Attenuated live virus	Humans	ELISpot	Up to 250 cells per 10^6 PBMC	See ref. ^81

Biological Advanced Research and Development Authority. BARDA Strategic Plan 2011-2016. 2011:1-20

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