Abstract
The potential for smallpox to be disseminated in a bioterror attack has prompted development of new, safer smallpox vaccination strategies. We designed and evaluated immunogenicity and efficacy of a T-cell epitope vaccine based on conserved and antigenic vaccinia/variola sequences, identified using bioinformatics and immunological methods. Vaccination in HLA transgenic mice using a DNA-prime/peptide-boost strategy elicited significant T cell responses to multiple epitopes. No antibody response pre-challenge was observed, neither against whole vaccinia antigens nor vaccine epitope peptides. Remarkably, 100% of vaccinated mice survived lethal vaccinia challenge, demonstrating that protective immunity to vaccinia does not require B cell priming.
1. Introduction
The CDC and NIH classify Variola major as a Category A pathogen because concerns remain that it, or closely related poxviruses, such as monkeypox, might be used to provoke fear and induce widespread morbidity and mortality in a bioterror attack [1,2]. Variola (Smallpox) is a particularly dangerous biological threat because of its clinical and epidemiologic properties [3,4]. Smallpox virus can be manufactured in large quantities, stored for an extended period of time, and delivered as an infectious aerosol. Vaccinia (Smallpox vaccine) has been used to protect against Smallpox but many individuals have not been vaccinated (the vaccine was no longer required in the U.S. after 1980), thus case-fatality rates could be higher than 25% of the population if smallpox were released as a bioterrorist weapon [2]. In addition, human monkeypox is an emerging zoonotic disease and potential biowarfare agent for which prophylactic agents are needed, and against which vaccinia (Smallpox) vaccination has been considered.
Mass vaccination, as was successfully implemented to eradicate smallpox worldwide, would be the logical course of protective action in response to deliberate dissemination of monkeypox and smallpox, but it poses a medical dilemma because the risks associated with vaccination using live-attenuated vaccinia are not negligible. The use of live attenuated vaccinia in immunization protocols where a significant percentage of the population is immunocompromised because of HIV infection, has raised some concern [5,6]. Data accumulated over the eradication campaign years showed that immunization with replication-competent, attenuated vaccinia was associated with serious adverse effects, such as encephalitis, vaccinia necrosum and eczema vaccinatum [7,8]. While their incidence was low at the time, today they could be significantly magnified because a greater proportion of the population is immunocompromised. Although the current US government stockpiled vaccine, ACAM2000, a vero-cell-culture derived vaccinia, has the advantage of limiting the risk of adventitious agents, the replicating virus has a similar adverse event profile compared to Dryvax [9]. As a result, development of safer smallpox vaccines has become a priority. Currently, modified vaccinia Ankara (MVA), a highly attenuated nonreplicating virus in mammalian cells, has a significantly limited adverse event profile and is currently in clinical trials [10].
The goal of our VennVax smallpox vaccine development program has been to demonstrate proof-of-principle that a genome-to-vaccine approach can be successfully applied to a potential bioterror agent. To develop VennVax, we systematically evaluated the vaccinia and variola genomes for conserved immunogenic HLA Class I and Class II epitopes and demonstrated that these epitopes possess properties essential to all successful vaccine antigens: 1) HLA binding and 2) ex vivo antigenicity in human subjects, 3) in vivo immunogenicity and 4) protection from lethal challenge. Previously, we reported immunoinformatic selection of 50 conserved and immunogenic variola/vaccinia Class II HLA epitope sequences, of which >80% were antigenic in ex vivo T cell assays performed with blood from Dryvax-exposed volunteers [11]. Here, we report that these T-cell epitopes are immunogenic and efficacious in an HLA transgenic mouse model of vaccinia infection when delivered as a heterologous DNA-prime/peptide-boost vaccine. Remarkably, vaccine-induced antibody production is not required for protection from challenge.
2. Methods
2.1 Multi-epitope DNA vaccine engineering.
Epitope sequences were concatenated to form two multi-epitope genes, each containing 25 HLA Class II epitopes that were identified by immunoinformatics methods, as described previously [10]. Initially, epitopes were assembled in a random sequence. To avoid creation of novel epitopes at epitope junctions, an algorithm which iteratively re-orders epitopes to reduce junctional immunogenicity (VaccineCAD) was used to optimize epitope order [12]. In addition, where re-ordering by VaccineCAD did not sufficiently reduce potential junctional immunogenicity, Gly-Pro-Gly-Pro-Gly spacer sequences were engineered between some epitopes to optimize epitope processing [13].
A Kozak sequence was engineered upstream of the coding sequence for efficient translation initiation. To target the immunogens to the Class II processing pathway, the tissue plasminogen activator (tPA) leader sequence (MQMSPALTCLVLGLALVFGEGSA) was placed upstream of epitope sequences to direct translation products to the secretory pathway. A histidine tag was incorporated downstream of the epitope sequences followed by two stop codons. Genes were synthesized by GeneArt and subcloned at pre-determined flanking restriction sites into pVAX1 (Invitrogen), a DNA vaccine vector that accommodates FDA recommendations for construction of plasmid DNA vaccines [14].
2.2 Plasmid DNA vaccine preparation.
High purity plasmids for immunizations were prepared by PureSyn, Inc. at pre-clinical grade. Each plasmid underwent quality control testing including spectrophotometric concentration and A260/A280 ratio determination (~1.9), restriction digest analysis to assure the presence of the multi-epitope genes, agarose gel electrophoresis determination of residual host RNA and DNA (none detected), and quantitative endotoxin testing (<24.9 EU/mg).
2.3 Peptide synthesis.
Peptides were manufactured using 9-fluoronylmethoxycarbonyl (Fmoc) chemistry by SynPep (Dublin, CA) and by New England Peptide (Gardner, MA). Master batch records indicate that peptides were purified to >80% as ascertained by analytical reversed phase HPLC and peptide mass was confirmed by MALDI-TOF mass spectrometry.
2.4 Peptide vaccine preparation.
The constituent peptides of the DNA vaccine were formulated in liposomes with immunostimulatory CpG oligodeoxynucleotide (ODN) 1826 (5′-TCCATGACGTT CCTGACGTT-3′; InvivoGen, San Diego, CA) [15]. Sterically stable cationic liposomes were prepared from three lipid components: dioleylphosphatidylethanolamine, dimethylaminoethanecarbamol-cholesterol, and polyethylene glycol 2000-phosphatidyl-ethanolamine (Avanti Polar Lipids, Alabaster, AL). The lipids were mixed, dried in a rotary evaporator and re-suspended in PBS to make empty multi-lamellar vesicles. These vesicles were then sonicated five times for 30 seconds each at 4°C to convert them into unilamellar liposomes. Unilamellar liposomes (10 nmol) were mixed with 1 mg/mL CpG ODN and peptides, flash frozen and freeze-dried overnight. To encapsulate CpG ODN and peptides in liposomes, the resulting powder was re-suspended with sterile distilled water and vortexed for 15 seconds every five minutes for 30 minutes at room temperature. PBS was added to yield a final liposome concentration of 10 mM lipid/mg ODN and peptides. Vesicles <150 nm in diameter were produced by 20–30 cycles of extrusion through polycarbonate filters using a Liposofast extruder (Avestin). Liposome formulations were prepared fresh for each study, one day before the first peptide immunization and stored at 4°C until a second peptide immunization two weeks later.
2.5 Mice.
HLA DR3 transgenic mice were obtained from Dr. Chella David (Mayo Medical School) under commercial license. The mice express the HLA DR3α and β genes on a B.10-Ab0 mouse Class II-negative background [16]. Experiments were conducted with mice 6 to 10 weeks old at the point of initiation. All studies were performed in full compliance with the standards of the University of Rhode Island and Saint Louis University Institutional Animal Care and Use Committees and in accordance with NIH publications entitled “Principles for Use of Animals” and “Guide for the Care and Use of Laboratory Animals.”
2.6 Vaccinations.
DNA-prime vaccine was administered to mice intramuscularly by needle stick injection with 50 μL of 50 μg naked DNA in sterile PBS injected into the quadriceps muscle of each leg. For peptide-boost immunizations, each mouse was anesthetized with ketamine/xylazine and administered a 30μl liposome preparation aliquot (50μg peptide) at 15 μl per nare, via micropipette.
2.7 ELISpot assay.
The frequency of epitope-specific splenocytes was determined by IFN-gamma ELISpot assay using the Mabtech IFN-gamma ELISpot Kit according to the manufacturer’s protocol (Mariemont, OH). Briefly, splenocytes were harvested from control and vaccinated mice. Pharmlyse (1X, BD Biosciences) was used to lyse red blood cells. The remaining lymphocytes were re-suspended in RPMI-10% fetal bovine serum-1% penicillin/streptomycin-1% L-glutamine-0.1% BME to a concentration of 4 × 105 cells/ml. Single cell splenocyte suspensions were transferred at 2.5×105/well to ELISpot plates pre-coated with anti-murine IFN-gamma by the manufacturer. Individual and pooled peptides were evaluated at 10 μg/ml in triplicate wells. Cells in RPMI 1640 media containing 10% FBS were also plated with a positive control Con A (2 μg/ml) or with no peptide as a negative control. ELISpot plates were incubated at 37°C, 5% CO2 for 2 days, incubated with a secondary HRP labeled anti-IFN-gamma antibody and developed by addition of TMB substrate. Spot counts were determined by Zellnet, Inc. using a Zeiss ELISpot plate reader. Results were recorded as the average number of spots over background and adjusted to spots per one million cells seeded. Responses are considered positive if the number of spots is: 1) at least twice average background, 2) greater than 20 spots forming cells per one million splenocytes over background (i.e. one response over background per 50,000 splenocytes), and 3) statistically significant by Student’s t-test in comparison with the corresponding spot forming cell data set for non-immunized mice (p<0.05).
2.8 Proliferation assay.
Splenocyte single cell suspensions were generated as described above. Peptides were diluted in RPMI to a final concentration of 20 μg/mL and Con A was diluted to 4 μg/mL. Equal volumes of cells and peptide or Con A were added to 96-well polystyrene tissue culture plates and incubated for 72 hours in a 37°C, 5% CO2 humidified incubator. 1 μCi of 3H-thymidine (MP Biomedical) was added to each well and incubated overnight. The proliferation assays were harvested with a Tomtec 96-well cell harvester and counted as cpm on a Wallac Trilux 1450 MicroBeta counter (Perkin Elmer). Student’s t-test was used for pairwise comparisons with p < 0.05 considered statistically significant.
2.9 Multiplex cytokine assay.
The Luminex 100 system and BioRad mouse 23-plex kits were used to assay IFNγ, IL-1β, IL-2, IL-10, IL-12p40, RANTES, MIP-1α and MIP-1β levels in peptide stimulated splenocyte cultures. Antibody-coupled beads specific for the listed cytokines were allowed to react with samples. After performing a series of washes to remove unbound protein, a biotinylated detection antibody specific for a different epitope on the cytokine was added to the beads. The reaction mixture was detected by addition of streptavidin-phycoerythrin (streptavidin-PE), which bound to the biotinylated detection antibodies. Results were read on the Luminex 100 system and analyzed using Masterplex software [17]. Unknown cytokine concentrations were automatically calculated using a standard curve derived from a recombinant cytokine standard. Student’s t-test was used for pairwise comparisons with p < 0.05 considered statistically significant.
2.10 Challenge inoculum preparation.
Vaccinia virus WR was prepared by sucrose cushion gradient ultracentrifugation. Briefly, infected cells were harvested and lysed with a dounce homogenizer in 10 mM Tris-HCl pH 9. The nuclei were pelleted and the cytosolic fraction sonicated then layered onto a 36% sucrose cushion in 10 mM Tris-HCl pH 9. The cushion/lysate was centrifuged at 32,900 x g for 80 minutes. The pelleted virus was re-suspended in 1 mM Tris-HCl, pH 9. The virus was titrated on the day of infection by making serial ten-fold dilutions of the stock virus in PBS and infecting BSC-1 cell monolayers in duplicate per dilution. The cultures were incubated for one hour in a 37°C, 5% CO2 humidified incubator and were overlaid with DMEM-5% fetal bovine serum, 1% carboxymethylcellulose. At 48 hours post-infection, monolayers were stained with crystal violet in 10% formaldehyde, plaques enumerated and the titer of the virus was calculated.
2.11 Respiratory challenge.
Mice were dosed with 10X LD50 vaccinia WR (~2.9 × 104 PFU) intranasally by the method used to administer the peptide vaccine. Mice were monitored daily for morbidity/mortality and weighed every other day.
2.12 Anti-vaccinia antibody titers.
Serum antibody levels were measured by standard indirect ELISA methods [18]. Antigen and control preparations were lysates from BSC-1 cells that were infected with multiplicity of infection (MOI)=0.1 or MOI=0 respectively using vaccinia virus WR and treated with psoralen under long wave UV light to inactivate the virus in the samples.
To determine the level of peptide-specific antibodies in serum samples, a direct anti-vaccinia virus ELISA was performed using a lysate from BSC-1 cells infected with VACV-WR. The clarified cell lysate was diluted in 50 mM carbonate-bicarbonate buffer pH 9.6 at 1:2500 and used to coat 96-well microtiter ELISA plates (Immulon-2 HB) at 4 °C overnight. Plates were blocked with PBS pH 7.2, 0.05% Tween20, 2% normal goat serum (Vector) at room temperature for 30 minutes. Mouse sera were added to wells at a 1:50 dilution in PBS pH 7.2, 0.05% Tween20 (PBS-T) and incubated for one hour at room temperature. Wells were washed with PBS-T and bound antibody was detected by incubation for one hour at room temperature with biotin-conjugated goat anti-mouse IgG (Caltag) at 1:2500 dilution. Plates were washed three times in PBS-T followed by the application of streptavidin-HRP (Zymed) at 1:4000 for thirty minutes. O-phenylenediamine dihydrochloride (0.4 mg/ml) in 50 mM citrate buffer (pH 5.0) and 0.05% hydrogen peroxide was added for fifteen minutes then the reaction stopped with 3N HCl. Optical density was measured at 490 nm. Student’s t-test was used for pairwise comparisons with p < 0.05 considered statistically significant.
3. Results
3.1 Multi-epitope DNA vaccine construction.
We designed two DNA vaccines, VennVax A and VennVax B, each of which contains a distinct set of 25 HLA class II epitopes (Figure 1, Table 1). The epitopes were previously identified using immunoinformatics methods that selected immunogenic and conserved sequences from 7 poxvirus genomes [11]. In those studies, 41 out of 50 Class II peptides induced T cell responses in Dryvax-immunized subjects; all 50 peptides including those confirmed in vitro using human PBMC were included in the VennVax vaccine constructs described here.
Figure 1. Multi-epitope DNA vaccine constructs.
Two synthetic genes (VennVax A and VennVax B), each encoding a distinct concatamer of 25 smallpox T-cell epitopes, were inserted individually into the pVAX1 DNA vaccine vector. Expression of the concatamers was directed for secretion by the tissue plasminogen activator (TPA) N-terminal signal sequence.
Table 1. VennVax T-cell Epitopes.
Column 1: peptide ID composed of four-digit ID followed by MHC class and abbreviated smallpox gene name. Epitope numbering was previously described in [12]. Column 2: amino acid sequence. Column 3: GenBank accession number of parent protein. Column 4: sequence location in protein.
| Peptide ID | Amino Acid Sequence |
M35027: AAA |
Location in Protein |
IFNγ ELISpot Response1 |
IFNγ ELISpot Response2 |
Proliferation2 | IFNγ2 | IL-22 | MIP-1a2 | MIP-1b2 | ||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| VennVax A | 4002_II_B1R | LDAVIRANNNRLPKRS | 48194.1 | 108-123 | + | POOL 1 | + | + | − | − | − | + |
| 4003_II_E2L | PEKLYLFKPRTVAPLDLIST | 48039.1 | 57-76 | − | ||||||||
| 4011_II_I2L | WGWYWLIIIFFIVLILLLLIYLYLKVVW | 48057.1 | 46-73 | − | ||||||||
| 4013_II_A20R | LKELLSLYKSLRFSDSAAIEKY | 48143.1 | 10-31 | + | ||||||||
| 4016_II_H1L | MDKKSLYKYLLLRSTGDMHKA | 48088.1 | 1-21 | − | ||||||||
| 4017_II_A24R | GVFYRPLHFQYVSYSNFILHRL | 48148.1 | 25-46 | − | POOL 2 | + | − | − | − | − | + | |
| 4018_II_L3L | GEMFVRSQSSTIIV | 48078.1 | 337-350 | + | ||||||||
| 4019_II_J3R | KLPYQGQLKLLLGELFFLSKL | 48083.1 | 33-53 | − | ||||||||
| 4020_II_L4R | LSIFNIVPRTMSKYELELI | 48079.1 | 42-60 | − | ||||||||
| 4026_II_J6R | YKYFIDLGLLMRMERKLSDKI | 48086.1 | 1252-1272 | − | ||||||||
| 4027_II_J6R | TGSQYYFSMLVARSQSTDIVC | 48086.1 | 727-747 | − | POOL 3 | − | − | − | + | − | − | |
| 4029_II_J6R | NDVDSNFVVAMRHLSLAGLLS | 48086.1 | 526-546 | − | ||||||||
| 4030_II_A32L | NLLKMPFRMVLTGGSGSGKTI | 48158.1 | 43-63 | − | ||||||||
| 4031_II_A4L | EIGLKSQESYYQRQLREQLARD | 48120.1 | 38-59 | − | ||||||||
| 4035_II_I8R | EFLHNYILYANKFNLTLPEDL | 48064.1 | 522-542 | + | ||||||||
| 4037_II_J6R | AGYKVNPTELMYILGTYGQQR | 48086.1 | 667-687 | + | POOL 4 | + | + | − | + | − | + | |
| 4039_II_G6R | SIIFINYTMSLTSHLNPSIEK | 48070.1 | 15-35 | + | ||||||||
| 4040_II_A26L | KFKTLNIYMITNVGQYILYIV | 48151.1 | 106-126 | + | ||||||||
| 4041_II_B18R | GYTALHYYYLCLAHVYKPGEC | 48217.1 | 168-188 | − | ||||||||
| 4042_II_J6R | GSIQDEIVAAYSLFRIQDLCL | 48086.1 | 456-476 | + | ||||||||
| 4043_II_G6R | GYLSAKVYMLENIQVMKIAAD | 48070.1 | 70-90 | + | POOL 5 | + | − | + | − | + | + | |
| 4046_II_F15L | PFHFQQPQFQYLLPGFVLTCI | 48034.1 | 83-103 | − | ||||||||
| 4048_II_J6R | SVNKFKFGAASTLKRATFGDN | 48086.1 | 1197-1217 | − | ||||||||
| 4049_II_A44L | SYDMFNLLLMKPLGIEQGSRI | 48175.1 | 255-275 | − | ||||||||
| 4050_II_I8R | SLPRIALVRLHSNTILKSLGF | 48064.1 | 226-246 | + | ||||||||
| VennVax B | 4000_II_D11L | GTNIWYSNSNRLMSINR | 48110.1 | 578-594 | − | POOL 6 | − | − | − | − | − | − |
| 4001_II_D6R | KKLLYLKFKTKETNRIYSI | 48105.1 | 504-522 | − | ||||||||
| 4004_II_A24R | ICDFVTDFRRRKRMGFFGN | 48148.1 | 545-563 | + | ||||||||
| 4005_II_A8R | GAVINQMVNTVLITVYEKLQLVIE | 48127.1 | 210-233 | − | ||||||||
| 4006_II_I5L | MQSLKFNRAVTIFKYIGLFIYIP | 48061.1 | 40-62 | + | ||||||||
| 4007_II_D5R | DTAVYRRKTTLRVVGTRKNPNCDT | 48102.1 | 170-193 | + | POOL 7 | − | − | − | + | − | − | |
| 4008_II_E6R | DADIVLNRHAITMYDKILSYIY | 48044.1 | 243-264 | + | ||||||||
| 4009_II_D12L | IDTMRIYCSLFKNVRLLKCVSDSWL | 48113.1 | 166-190 | − | ||||||||
| 4010_II_E8R | IYNILFWFKNTQFDITKH | 48047.1 | 136-153 | − | ||||||||
| 4012_II_B15R | LTEYIYWSSYAYRNRQCAGQLYS | 48212.1 | 30-52 | − | ||||||||
| 4014_II_A23R | NQPWIKTISKRMRVDIINHSIVT | 48147.1 | 168-190 | + | POOL 8 | + | + | − | + | − | − | |
| 4015_II_L3L | LVRSRKAVGFPLLKAAKRISHGSM | 48078.1 | 109-132 | − | ||||||||
| 4021_II_A18R | VSEVVSNMRKMIESKRPLYITLH | 48140.1 | 90-112 | + | ||||||||
| 4022_II_J3R | FYNLGMIIKWMLIDGRHHDPIL | 48083.1 | 82-103 | + | ||||||||
| 4023_II_A7L | GDDIVRLRTTSDIIQFVN | 48124.1 | 526-543 | − | ||||||||
| 4024_II_L2R | RPLIRLFIDILLFVIVIYIF-TVRLVSRNYQMLLAL | 48077.1 | 38-72 | − | POOL 9 | + | + | − | + | − | − | |
| 4028_II_C10L | PVTEDDYKFLSRLVLYAKSQS | 47986.1 | 279-299 | − | ||||||||
| 4032_II_N2L | VSILNKYKPVYSYVLYENVLY | 48002.1 | 140-160 | − | ||||||||
| 4033_II_D10R | NKFFEVIFFVGRISLTSDQII | 48109.1 | 171-191 | + | ||||||||
| 4034_II_D10R | SSIISQIIKYNRRLAKSIICE | 48109.1 | 6-26 | − | ||||||||
| 4036_II_J6R | ASNQVKFYFNKRLNQLTRIRQ | 48086.1 | 330-350 | − | POOL 10 | − | − | − | − | − | − | |
| 4038_II_F12L | YETIEILRNYLRLYIILARNE | 48029.1 | 15-35 | − | ||||||||
| 4044_II_D4R | DKFFIQLKQPLRNKRVCVCGI | 48100.1 | 47-67 | − | ||||||||
| 4045_II_G8R | VFYRGAENIVFNLPVSKVKSC | 48074.1 | 57-77 | + | ||||||||
| 4047_II_J6R | KNNMIRSYIVARRKDQTARSV | 48086.1 | 269-289 | + |
Epitope sequences were randomly concatemerized, at first. To avoid production of neo-epitopes at epitope junctions, the VaccineCAD algorithm was used to re-arrange epitopes in an order that diminishes potential junctional immunogenicity. The default order for each construct contained significant predicted immunogenicity at a single junction in VennVax A and four junctions in VennVax B (EpiMatrix scores >10). Re-ordering of epitopes by VaccineCAD and insertion of GPGPG spacers [19] produced sequences with minimized junctional immunogenicity (EpiMatrix scores < 0.2) (Supplemental Data Tables 1 and 2).
3.2 Immunogenicity of individual vaccines in HLA DR3 mice.
Mice were twice injected intramuscularly with one of two DNA vaccines each encoding 25 DR epitopes (VennVax A or VennVax B) over a two-week interval. Two weeks later, they were then boosted twice intranasally with corresponding epitope peptides formulated in liposomes with immunostimulatory CpG ODN 1826 over a two-week interval. A control group of mice received empty vector in the DNA-prime phase and peptide-free liposomes in the boost phase. Two weeks following the final immunization, splenocytes were isolated and pooled to measure T cell responses to individual epitopes by IFNγ ELISpot. Immunization of DR3 transgenic mice stimulated statistically significant (Student’s t-test, p<0.01) T cell responses to 10 of 25 VennVax A epitopes (40%): 4002_II_B1R, 4013_II_A20R, 4018_II_L3L, 4035_II_I8R, 4037_II_J6R, 4039_II_G6R, 4040_II_A26L, 4042_II_J6R, 4043_II_G6R and 4050_II_I8R (Figure 2, top). For VennVax B immunized mice, 7 of 25 epitopes (28%) were immunogenic with statistical significance at p<0.01: 4004_II_A24R, 4006_II_I5L, 4014_II_A23R, 4021_II_A18R, 4033_II_D10R, 4038_II_F12L and 4047_II_J6R; 3 of 25 epitope (12%) were immunogenic with statistical significance at 0.01<p<0.05: 4008_II_E6R, 4022_II_J3R and 4045_II_G8R (Figure 2, bottom). 4034_II_D10R elicited >50 spot forming cells per million splenocytes but the result did not meet statistical significance. Also noteworthy are significant responses observed for the pools of VennVax A and B epitopes among mice respectively immunized.
Figure 2. Cell-mediated response to immunization of HLA DR3 transgenic mice with VennVax A or VennVax B epitopes.
Mice were primed with plasmid DNA vaccine and boosted with peptides comprising the VennVax A or VennVax B epitope sets or vaccine vehicle containing no epitopes. Epitope-specific cellular responses in splenocyte cultures for the VennVax A (top) and VennVax B (bottom) epitopes were measured by IFNγ ELISpot. Data are the mean spot forming cells (SFC) per million splenocytes ± standard deviation derived for 5 mice treated comparably. Individual epitope and pooled epitope responses in vaccinated mice showing statistical significance (Student’s t-test) when compared with controls are indicated: * p<0.05, ** p<0.01.
3.3 Immunogenicity of combined vaccines in HLA DR3 mice.
Because breadth of T cell response is important for efficacious vaccine responses [20,21], we sought to increase the number of reactive epitopes in a single vaccine by combining the two sets of epitopes. DR3 mice were co-immunized with the VennVax A and VennVax B epitope sets using the DNA-prime/peptide-boost strategy described above and then challenged with vaccinia WR. DNA was delivered by needle stick injection, intramuscularly, twice, each dose two weeks apart, followed two weeks later by peptide boosting intranasally, twice, each dose two weeks apart, with the corresponding epitope peptides formulated in liposomes with CpG ODN 1826. Both DNA and peptide vaccinations were administered at doses identical to those delivered in the first study i.e. VennVax A and VennVax B were delivered at half the dose used in the single vaccine study. In parallel, a non-immunized group of mice was followed to control for vaccine-specific responses. These mice received empty vector in the DNA-prime phase and peptide-free liposomes in the boost phase.
To assure that vaccination was successful, three mice from each group were euthanized before vaccinia challenge and vaccine-specific immunogenicity was measured using pooled peptide stimulation of splenocytes from individual mice in IFNγ ELISpot (Figure 3, top), proliferation (Figure 3, middle) and multiplex ELISA (Figure 3, bottom) assays. Immunization stimulated significant T cell responses to 6/10 peptide pools by IFNγ ELISpot (Student’s t-test, p<0.01). Peptide pools that elicited responses contained epitopes that were observed to be immunogenic as individual peptides in the previous study. Pools 3, 7 and 10 produced no significant IFNγ secretion although they contained peptides that elicited positive responses in the previous study. Borderline responses in the first study may explain this discrepancy.
Figure 3. Cell-mediated response to co-immunization of HLA DR3 transgenic mice with VennVax A and VennVax B epitopes.
Mice were primed with plasmid DNA vaccine and boosted with peptide pools comprising the VennVax A and VennVax B epitope set or vaccine vehicle containing no epitopes. Splenocyte responses to pooled epitopes were measured by IFNγ ELISpot (top), proliferation (middle) and multi-cytokine ELISA (bottom). ELISpot data are the mean spot forming cells (SFC) per million splenocytes for individual (circles) and groups (bars) of mice. Proliferation data are stimulation indices over background response for individual (circles) and groups (bars) of mice. The data labels “1-5” and “6-10” refer to separate stimulations with larger pools containing 5 of the smaller pools. Pooled epitope responses in vaccinated mice showing statistical significance (Student’s t-test) when compared with controls are indicated: * p<0.05, ** p<0.01.
Significant peptide-stimulated proliferation was observed for 4/10 pools (Student’s t-test, p<0.01) and was consistent overall with IFNγ ELISpot responses. Significant IFNγ, IL-2 and MIP-1α/β production (p<0.05) was observed for peptide pools that were positive in IFNγ ELISpot. Importantly, the multi-cytokine Th1 vaccine-induced response suggests that T cells with robust effector and memory potential may have been generated. The overall trend of IFNγ responses across peptide pools observed by ELISA is similar to the ELISpot data, though the stimulation indices are not as high as might be expected. This difference may be explained by the fact that the ELISA and ELISpot assay measure different properties of cytokine production. While ELISAs measure bulk levels of cytokine secretion, ELISpot assays detect frequencies of cytokine-producing cells. Studies have demonstrated that the ELISpot assay is significantly more sensitive than ELISA, meaning that cells producing cytokine at levels that are not detected by ELISA may be observed by ELISpot assay [22,23]. Moreover, cytokine secretion levels as measured by ELISA may be diminished because cytokine is degraded or consumed through binding to cytokine receptors in the supernatant or on the surface of nearby cells. In contrast, the ELISpot method captures cytokine directly upon release from the cell by plate-bound antibody.
No antibody response was observed in sera taken from vaccinated mice before challenge in indirect ELISAs performed using vaccinia lysate (Figure 4, top) and epitope peptides as antigens. Because no response was observed in non-immunized mice as well, and no statistically significant difference between the two groups was determined by Student’s t-test, we conclude that the T-cell epitopes in this formulation of VennVax contain no B-cell epitopes or, alternatively, induction of antibodies was inefficient.
Figure 4. Pre- and post-challenge antibody responses in HLA DR3 transgenic mice co-immunized with VennVax A and VennVax B epitopes.
Antibody ELISAs using vaccinia lysate as antigen were performed using sera from immunized and control mice before and after vaccinia challenge (105 days post-infection). Pre-challenge ELISA (top panel) shows that the VennVax A and VennVax B epitopes do not elicit antibody production; no statistically significant difference is observed between vaccine and control groups by the Student’s t-test. Post-challenge (bottom panel), surviving control mice developed higher antibody titers with statistical significance by the Student’s t-test (p<0.05).
3.4 In vivo lethal challenge.
Next, we set out to evaluate vaccine efficacy against lethal vaccinia challenge in DR3 transgenic mice. Four weeks following the final immunization, mice were challenged intranasally with 10X LD50 of vaccinia WR (~2.9 × 104 PFU, as determined in preliminary LD50 and infectivity measurements for the DR3 strain; data not shown). An intranasal challenge was chosen because the respiratory route is the most likely course of smallpox infection in a bioterror event. Remarkably, 100% of vaccinated mice (N=18) survived lethal vaccinia challenge while only 19% of control mice (N=16) recovered (Student’s t-test, p<0.001) (Figure 5, top). Vaccinated mice showed minimal signs of illness as weight loss <5% of pre-challenge weight was observed during the challenge period (Figure 5, bottom). In contrast, the large majority of non-immunized mice steadily lost weight and died; only three mice regained weight after initial weight loss and survived. All surviving mice lived >3 months post-infection.
Figure 5. Epitope-driven vaccine induces protection against lethal vaccinia challenge in HLA DR3 transgenic mice.
Mice were co-immunized with VennVax A and VennVax B and boosted with corresponding peptides in liposomes. (Top) Survival rate of mice after challenge with 10X LD50 vaccinia WR. (Bottom) Percent change in body weight of mice after challenge.
3.5 Post-challenge immunogenicity analysis.
To measure the influence of epitope-driven vaccination on the immune response to challenge, surviving mice from each arm were sacrificed >25 weeks post-challenge and their antibody and T cell responses assayed. A significant antibody response was observed among vaccinated mice and placebo mice that survived (Figure 4, bottom). The average antibody response among vaccinated mice was lower than in surviving control mice and is statistically significant at multiple serum dilutions (Student’s t-test, p<0.05).
Vaccine specific immunogenicity was measured using individual peptide stimulation of splenocytes from individual mice in IFNγ ELISpot assays. Epitopes from peptide pools that gave rise to positive responses in the pre-challenge ELISpot assay were assayed here. Mice that were administered vaccine demonstrated stronger and broader immune responses to VennVax epitopes than control mice while both groups responded comparably to vaccinia lysate stimulation (Figure 6).
Figure 6. Post-challenge cell-mediated response in HLA DR3 transgenic mice co-immunized with VennVax A and VennVax B epitopes.
Epitope-specific cellular responses in splenocyte cultures were measured by IFNγ ELISpot. Data are the mean spot forming cells (SFC) per million splenocytes ± standard deviation derived for 3 control and 3 vaccinated mice. Individual epitope responses in vaccinated mice showing statistical significance (Student’s t-test) when compared with controls are indicated: * p<0.05, ** p<0.01.
4. Discussion
The findings presented here build on our earlier work using the EpiMatrix epitope mapping algorithm to systematically evaluate seven vaccinia and variola genomes for conserved immunogenic HLA Class I and Class II epitopes with high potential for promiscuous immunogenicity. We demonstrated that the predicted epitopes bind HLA and are largely antigenic in ex vivo studies of ACAM2000-vaccinated human subjects [11]. Here, we applied those results to develop an epitope-driven vaccine allowing us to demonstrate that these conserved variola/vaccinia HLA Class II epitopes can be administered in a DNA-prime, peptide-boost multi-epitope vaccine that is both broadly immunogenic and protective in vivo.
4.1 Cell mediated response.
The 50 Class II promiscuous epitopes in VennVax were derived from 37 distinct proteins, of which 29 were independently reported to be antigenic in T cell assays [24,25,26], as were the others as we previously described [11]. Thus, the vaccine epitopes included sequences from antigens that are processed and presented in vaccinia infection. We found that 17 epitopes were immunogenic in vivo in HLA DR3 transgenic mice. These results are consistent with previous studies that showed strong antigenicity for these epitopes in Dryvax vaccinees [11]. In immunogenicity studies performed in HLA DR1 mice using the VennVax A vaccine, six of 25 (24%) epitopes were immunogenic according to the same statistical significance standard applied in the HLA DR3 mouse study (p<0.01): 4017_II_A24R, 4027_II_J6R, 4030_II_A32L, 4041_II_B18R, 4043_II_G6R and 4050_II_I8R (Supplemental Figure 1). As the vaccine epitopes were predicted to cover a HLA diverse population, not only DR1 or DR3 carriers, it comes as no surprise that not every epitope tested positive for immunoreactivity in these specific HLA transgenic mouse strains. In fact, of the observed responses, 58% of VennVax A and B epitopes correlated with the predictions made by EpiMatrix for the DR3 allele, i.e. 58% of epitopes were correctly predicted to stimulate or to not stimulate immune responses. Of the 42% that did not correlate, 15 were predicted to bind to this MHC allele but were non-immunogenic. These results are not unforeseen because in vivo there are many more factors beyond binding to MHC (e.g. plasmid uptake, expression, peptide processing) that impact immunogenicity. Even so, we found that two epitopes were reactive in both the HLA DR1 and DR3 strains, 4043_II_G6R and 4050_II_I8R (a published DR1 epitope [26]), illustrating that these sequences are immunologically promiscuous and therefore make good epitope-driven vaccine immunogens.
Remarkably, as many as 14 of these epitopes contributed to protective efficacy against lethal vaccinia challenge in DR3 transgenic mice, as measured by antigen-specific recall post-challenge (Figure 6). The results of this study are consistent with previous findings regarding breadth of immune response to vaccinia [11,27]. Importantly, although control mice responded to vaccinia antigens with a T cell response that was not focused on the vaccine epitopes, vaccinated mice exhibited long term recall responses to vaccine immunogens as expected. These results suggest that the memory responses in vaccinated and control mice are composed of different vaccinia-specific T cell repertoires. As the vaccine focused repertoire provided protection, this study demonstrates the power of immunoinformatics to tease out the essential information needed to design an efficacious vaccine from large genome datasets. Specifically, for the murine study carried out under these conditions, immune response to 14 epitopes appears to have been sufficient for protection against a lethal aerosol challenge.
4.2 Humoral response.
Conventional thinking is that B cells recognize conformational epitopes on structurally intact protein antigens to give rise to robust, high affinity antibody responses while short linear peptides are intrinsically structurally unstable and make poor B cell immunogens. However, peptides may adopt sufficiently stable structures to raise antibodies not only to the immunizing peptides themselves but also to corresponding parts in their native proteins [28,29]. Hence, we set out to determine if B cells contributed to the observed vaccine efficacy by measuring vaccine-induced antibody levels prior to challenge. The absence of a pre-challenge antibody response demonstrated that primed B cells are unnecessary for vaccinia protection, a remarkable observation because of the importance of antibody response ascribed to vaccine-mediated protection from vaccinia and other pathogens, in general [30,31]. This result is consistent with vaccinia challenge of MHC Class I epitope immunizations that provided protection in wild-type and HLA transgenic mice [32,33]. Notably, these studies involved co-immunization with an (unrelated i.e. non-cognate) MHC Class II epitope, as robust CD8 T cell proliferation and function are dependent on CD4 T cell responses [34]. In the present study, we demonstrate that immunization with MHC Class II epitopes alone is sufficient to provide protection. Our finding is further supported by a study showing that MVA vaccination protected B cell deficient mice from lethal vaccinia challenge [35]. Moreover, the same study showed a similar result for CD8 T cell deficient mice but poor protection in CD4 T cell and MHC Class II deficient mice, reflecting the importance of MHC Class II epitopes, such as those in VennVax, to vaccine efficacy. Our results are consistent with the findings from this prior vaccine challenge study and with those of a similar study of vaccinia infection [36].
Post-challenge, the average antibody response among vaccinated mice was lower than in surviving control mice. On the surface, it is surprising that vaccination with HLA Class II epitopes does not confer an advantage in terms of enhanced bulk antibody response. However, it can be argued that the immune response to challenge in vaccinated mice was focused, while non-immunized mice mounted an unfocused response that provided a small minority the capacity to overcome challenge while such a response could not confer protection on the large majority. Control survivors, over the course of illness, may have processed and presented an adequate set of T-cell epitopes that helped activate an antibody response capable of preventing death. Mice that died may not have selected an ensemble of epitopes that could activate the needed antibody response. Put another way, we hypothesize that at a higher challenge dose, all non-immunized mice would die without sufficient opportunity to identify a protective set of epitopes, but vaccinated mice mounting a focused response would live. Alternatively, antibody responses in the placebo-treated mice that did not survive may not have been sufficiently high to protect them from infection. If their responses could be measured, the average placebo response would probably be significantly lower than was observed in vaccinated mice. Finally, it is also possible that the lower antibody response in vaccinated mice is due to a lower antigenic load resulting from vaccine-mediated reduction in viral replication.
4.3 Epitope-driven vaccines.
There remains an unmet need for strategies that would provide a safer, more effective smallpox vaccine that does not require exposure to live virus. Moreover, the closely related monkeypox virus is considered an emerging zoonotic pathogen, particularly in Africa where a large percentage of the population is immunocompromised by HIV infection [37]. Conservation amongst poxviruses would suggest that vaccines that are designed for smallpox can be used to make a monkeypox vaccine, as well. In VennVax, we found that 48 out of the 50 HLA Class II epitopes are >90% identical and 68% of epitopes are completely identical in Monkeypox Virus Zaire 79. Strong conservation of vaccine epitopes in monkeypox virus suggests that VennVax could also be developed as a monkeypox vaccine.
Genome-derived epitope-driven vaccines such as VennVax may have a significant advantage over conventional vaccines, as the careful selection and construction of the immunogenic components may diminish undesired side effects such as have been observed with whole pathogen and protein subunit vaccines. It is noteworthy that a few epitope-driven vaccines against viral and microbial pathogens have reached the stage of Phase I or II clinical trials in humans. For example, Bionor Immuno’s HIV p24 gag peptide vaccine (Vacc-4X) was demonstrated to be safe and well-tolerated in Phase I trials [38] and dose-dependent and immunogenic in Phase II trials in Norway [39]. In the cancer vaccine field, where the concept of epitope-driven vaccines is well-established, many more peptide vaccines have successfully passed preclinical tests and entered into Phase I/II clinical trials [40]. Experimental validation needed to push forward these vaccines into clinical trials is now emerging and promises to enable epitope-driven vaccines to claim a more prominent place in the vaccine world.
As an alternative to the stand-alone approach, epitope-driven vaccines may be combined with other modalities in prime-boost immunizations. For example, while MVA is safer than ACAM2000, it requires higher and more doses to achieve efficacy [35] thus limiting the number of doses available when resources are limited and demand is high. Priming MVA vaccination with VennVax could be a dose-sparing strategy that would expand the potential of the US government’s MVA stockpile to greater numbers of immunocompromised persons.
In conclusion, this study demonstrates that VennVax provides excellent protection against lethal respiratory vaccinia challenge in a HLA transgenic mouse model. To our knowledge no HLA Class II epitope-driven subunit vaccine for smallpox has achieved a comparable level of protection in this challenge model. Importantly, we clearly illustrate that a genome-derived epitope-driven vaccine provides protection from a bio-terror pathogen, like we have shown previously for F. tularensis [41,42]. We are now poised to further develop VennVax to prime MVA vaccination as a dose-sparing strategy to expand the supply of smallpox vaccine.
Supplementary Material
Acknowledgements
The authors wish to acknowledge the efforts of Matthew Ardito, who helped in the bioinformatics analysis and Drs. David Weiner and Michele Kutzler for performing the DR1 mouse study. We also thank Tobias Cohen and Elizabeth McClaine for their assistance with manuscript editing and graphics production. This study was supported by the National Institutes of Health grant 5R43AI058376-02 (ADG).
Footnotes
Conflict of interest statement
Two of the contributing authors, Anne S. De Groot and William D. Martin are senior officers and majority shareholders at EpiVax, Inc., a privately-owned vaccine design company located in Providence, RI. Dr. De Groot is also a faculty member at the University of Rhode Island and Brown University. These authors acknowledge that there is a potential conflict of interest related to their relationship with EpiVax and attest that the work contained in this research report is free of any bias that might be associated with the commercial goals of the company.
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