As I posted earlier the VLP tech makes it easy to test a vaccine quickly and the Technosphere makes it easy to distribute a vaccine without refrigeration or injection.
Many companies work on VLP tech nut that I am aware only Mannkind has a well studied and proven way of administering VLP by inhalation (no needles or health care workers needed) and no refrigeration.
If I were a health official looking at rapidly testing and then mass distributing a vaccine I would certainly at least look into this option.VLP text is the basis for at least one vaccine out there (the Canadian one now being tested I thing). And it seems to work in non human primates according to the reference I provide below. . If I was an living next to an Ebola hotspot I certainly would be more then happy to participate in a clinical trial...
JPG
jid.oxfordjournals.org/content/196/Supplement_2/S430.longEbola Virus-Like Particle-Based Vaccine Protects Nonhuman Primates against Lethal Ebola Virus Challenge
Kelly L. Warfielda, Dana L. Swensona, Gene G. Olinger, Warren V. Kalina, M. Javad Amanb and Sina Bavari
+ Author Affiliations
US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland
+ Author Notes
↵b Present affiliation: Integrated BioTherapeutics Inc., Frederick, Maryland.
Reprints or correspondence: Dr. Sina Bavari or Dr. Kelly Warfield, US Army Medical Research Institute of Infectious Diseases, 1425 Porter St., Fort Detrick, MD 21702 (sina.bavari@us.army.mil or kelly.warfield@us.army.mil).
Presented in part: Filoviruses: Recent Advances and Future Challenges, International Centre for Infectious Diseases Symposium, Winnipeg, Manitoba, Canada, 17–19 September 2006.
↵a K.L.W. and D.L.S. contributed equally to this work.
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Abstract
Background. Currently, there are no licensed vaccines or therapeutics for the prevention or treatment of infection by the highly lethal filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), in humans.We previously had demonstrated the protective efficacy of virus-like particle (VLP)-based vaccines against EBOV and MARV infection in rodents.
Methods. To determine the efficacy of vaccination with Ebola VLPs (eVLPs) in nonhuman primates, we vaccinated cynomolgus macaques with eVLPs containing EBOV glycoprotein (GP), nucleoprotein (NP), and VP40 matrix protein and challenged the macaques with 1000 pfu of EBOV.
Results. Serum samples from the eVLP-vaccinated nonhuman primates demonstrated EBOV-specific antibody titers, as measured by enzyme-linked immunosorbent assay, complement-mediated lysis assay, and anti-body dependent cell-mediated cytotoxicity assay. CD44+ T cells from eVLP-vaccinated macaques but not from a naive macaque responded with vigorous production of tumor necrosis factor-α after EBOV-peptide stimulation. All 5 eVLP-vaccinated monkeys survived challenge without clinical or laboratory signs of EBOV infection, whereas the control animal died of infection.
Conclusion. On the basis of safety and efficacy, eVLPs represent a promising filovirus vaccine for use in humans.
During the past 20 years, owing to advances in molecular biology and in the understanding of basic virology, scientists have been able to develop subunit vaccines based on virus-like particles (VLPs). VLPs for many viruses have been developed and are based on the knowledge that expression of specific viral structural proteins results in the self-assembly of particles that morphologically resemble the authentic virus [1]. Some of the many advantages of using VLPs as vaccines include (1) their similar morphology to the live enveloped or nonenveloped viruses from which they are derived; (2) a strong safety profile, since they are nonreplicating; (3) no concerns regarding viral vector or preexisting antivector immunity; (4) the fact that they can be generated in large quantities by use of mammalian or insect cell lines; (5) their ability to generate innate, humoral, and cellular immunity; and (6) the fact that they have been safely and effectively administered in humans [1–7].
We and others have demonstrated previously the generation of enveloped Ebola VLPs (eVLPs) in mammalian and insect cell-expression systems [8–13]. VLPs containing glycoprotein (GP) and VP40 derived from Ebola virus (EBOV) have been used successfully to vaccinate rodents [2, 13–17]. Both BALB/c and C57BL/6 mice have been protected against a range of challenge doses (⋃10–1,000 pfu or ⋃300–30,000 LD50) by means of dose-dependent eVLP vaccination in the presence or absence of adjuvant [2, 14, 15]. Addition of saponinderived QS-21 or RIBI adjuvant to the eVLP-vaccine regimen allows administration of a decreased dose of the vaccine and completely protects mice and guinea pigs against challenge, even after only 1 inoculation (authors' unpublished data) [18]. eVLP vaccination completely prevents viremia and clinical symptoms after EBOV challenge in rodents but does not induce sterile immunity, as evidenced by an expansion of immune responses to viral proteins not present in the vaccine, after challenge [2, 14, 15, 18]. However, the question of whether eVLPs would be able to protect nonhuman primates against EBOV infection has remained. Therefore, the goal of the current study was to determine whether eVLPs would be viable vaccine candidates for the protection of primates against lethal EBOV challenge.
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Materials and Methods
Generation and characterization of eVLPs. 293T-derived eVLPs containing EBOV GP, nucleoprotein (NP), and VP40 were prepared essentially as described elsewhere [9, 14, 16, 17]. Total protein concentrations in the vaccine preparations were determined in the presence of detergent, by use of a Bradford protein assay (BioRad). Expression of GP, NP, and VP40 in each vaccine preparation was verified by Western blot analysis [9, 17, 18]. eVLPs were processed and imaged via electron microscopy, as described elsewhere [9, 14, 16, 17]. Endotoxin levels in all eVLP preparations used in this study were <0.03 endotoxin units/mg, as determined by the Limulus amebocyte lysate test (Invitrogen).
Animals. In testing before the start of this study, the cynomolgus macaques used were found to be antibody negative for filovirus, simian T cell leukemia virus—1, simian immunodeficiency virus, and herpes B virus. The eVLP-vaccinated monkeys received 3 intramuscular injections, at 42-day intervals, of ⋃1.0 mL of sterile saline containing 250 µg of eVLPs and 0.5 mL of RIBI adjuvant (Corixa). Blood samples were obtained from the femoral vein of monkeys under anesthesia. Female cynomolgus macaques of 3–4 kg in weight were challenged by intramuscular injection of ⋃1000 pfu of Zaire EBOV (ZEBOV; 1995 outbreak strain) [19]. Viremia was determined by means of a traditional plaque assay [20]. Hematology and kidney- and liver-associated enzymes were assessed as described elsewhere [21]. For ethical reasons, the use of relevant historical control animals was required by the Laboratory Animal Care and Use Committee of the US Army Medical Research Institute of Infectious Diseases (Fort Detrick, MD), to reduce the number of nonhuman primates needed in these studies. For this reason, the control monkey in the current study was not treated, so that, for the data analysis, results for this monkey could be combined with those for 23 historical control animals challenged with the same seed stock of ZEBOV at the same dose and via the same route.
All EBOV-infected animals and their samples were handled under maximum containment in a biosafety level 4 laboratory at the US Army Medical Research Institute of Infectious Diseases (Fort Detrick, MD). Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adhered to principles stated in the Guide for the Care and Use of Laboratory Animals [22]. The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (Rockville, MD).
Total antibody responses against EBOV. Levels of EBOVspecific antibodies were determined from serumor plasma samples by use of ELISA, as described elsewhere [14]. Antibody titers were defined as the reciprocal of the highest dilution giving a net optical density value ⩾0.2.
Determination of complement-mediated lysis by EBOV GP-specific antibody. Antibodies recognizing viral antigens on infected cells can bind complement via their Fc region and can initiate activation of the complement cascade, resulting in killing of the virus-infected cell. To determine whether eVLP vaccination generated antibodies capable of inducing complementmediated lysis, Vero cells were infected at an MOI of 10 with EBOV GP-expressing Venezuelan equine encephalitis virus (VEE) replicons, produced as described elsewhere [23], and were incubated in a humidified, 5% CO2 incubator at 37°C for 16 h. After incubation, Vero cells were removed from the plate by means of trypsinization and were labeled with 100 µCi of 51Cr for 1 h. Cells were washed 3 times in RPMI 1640 without fetal bovine serum (FBS) and were resuspended to 100,000 cells/mL in RPMI 1640 containing 10% FBS. Next, 100 µL of Vero cells were plated in each well of a 96-well plate containing various dilutions of plasma samples from eVLP-vaccinated and control monkeys. Then, low-endotoxin guinea pig complement was added, at a final dilution of 1:20, and samples were incubated for 3 h. The amount of radioactivity released into the supernatants was determined with a γ-radiation counter. Spontaneous lysis was measured in VEE replicon-expressing EBOV GP-infected cells [23] with complement added but no antibodies present, and total lysis was measured in Vero cells incubated with 1% Triton X-100. Percentage of specific lysis was calculated as [(experimental release—spontaneous release)/(maximum release—spontaneous release)]×100.
Antibody-dependent cell-mediated cytotoxicity (ADCC) assay. ADCC occurs when virus-specific antibodies coat target infected cells and make them vulnerable to killing during coculture with other cytolytic cells. To determine whether antibodies stimulated by eVLP vaccination are capable of inducing ADCC, Vero cells were infected with VEE replicon—expressing ZEBOV GP for 16 h and were labeled with 100 µCi of 51Cr, as described above. Labeled Vero cells were plated in a 96-well plate (10,000 cells/well). Primate plasma from previously vaccinated or naive (negative control) animals was added at various dilutions to labeled, GP-expressing Vero cells. Purified effector cells (peripheral blood mononuclear cells [PBMCs]), resuspended in RPMI 1640 with 10% FBS, were added to antibody-coated target cells at the following effector-to-target cell (ET) ratios: 1:10, 1:20, 1:40, and 1:80. Optimization of the assay was determined by choosing the ET ratio that produced the least background in wells with no antibody or with plasma from unvaccinated animals. Each plate was incubated for 4 h at 37°C in the presence of 5% CO2. After 4 h, centrifugation of each plate was done at 250 g to pellet the cells, 50 µL of supernatant was removed, and 51Cr released into supernatant was quantified by use of a γ-radiation counter. Spontaneous lysis was measured in VEE replicon-expressing EBOV GP-infected cells [23] with effector cells added but no antibodies present, and total lysis was measured in Vero cells incubated with 1% Triton X-100. Percentage of specific lysis was calculated as [(experimental release—spontaneous release)/(maximum release—spontaneous release)]×100.
T cell responses to EBOV peptides in eVLP-vaccinated nonhuman primates. Epitopes recognized by circulating CD4+ and CD8+ T cells were determined as described elsewhere [15, 24], with several minor modifications. In brief, EBOV-specific responses were analyzed by culturing PBMCs with 1–5 µg of overlapping 15-residue peptides representing EBOV GP, NP, or VP40 (Mimotopes) or 1 µg/mL staphylococcal enterotoxin B (SEB) in complete RPMI 1640 containing 10 µg/mL brefeldin A. After 18 h of culture, the cells were stained with anti-CD44, -CD8, or -CD4 (Pharmingen) in brefeldin A. After cell-surface staining, cells were fixed in 1% formaldehyde, permeabilized with saponin, and stained with anti-tumor necrosis factor (TNF)-α phycoerythrin (Pharmingen). Samples with an increase in the frequency of TNF-α-positive cells of >2-fold above background, as assessed by no peptide stimulation or by response to an irrelevant peptide (Lassa virus N: RPLSAGVYMGNLSSQ), were considered to be positive.
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Results
Humoral responses to EBOV infection, after eVLP vaccination. To determine whether eVLP vaccination elicits humoral responses in nonhuman primates, total antibody responses in blood from the eVLP-vaccinated macaques were determined, by ELISA using irradiated whole EBOV virions, immediately before each vaccination and before challenge (figure 1A). Total EBOV-specific antibodies in the eVLP-vaccinated macaques rose 3–10-fold after the first vaccination and plateaued after 2 vaccinations (figure 1A).
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Figure 1
Antibody responses in nonhuman primates vaccinated with Ebola virus-like particles (eVLPs) containing glycoprotein (GP), nucleo-protein, and VP40. Cynomolgus macaques were vaccinated 3 times, at 6- week intervals, with 250 µg of eVLPs in RIBI adjuvant. A, Total anti- Ebola virus (EBOV) antibody titers for individual animals (VLP1-5), as determined by ELISA. B, Induction of complement-mediated lysis indicated in plasma samples from eVLP-vaccinated macaques. EBOV GP-expressing Vero cells were incubated with guinea pig complement and the indicated dilutions of plasma. The percentage lysis was determined as compared with that in untreated cells. C, Results of lysis of EBOV GP-expressing target cells by an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. Target cells incubated with plasma from eVLP-vaccinated macaques or an unvaccinated animal (naive) and human effector cells (effector-totarget cell ratio, 40:1) showed various levels of ADCC antibody titers. Error bars indicate SDs.
Neutralizing titers were observed in the eVLP-vaccinated macaques, consistent with our results in previous studies of rodents [14, 16], with 80% plaque reduction/neutralization titers ranging from 1:20 to 1:160 (data not shown). Additional studies of antibody function revealed that eVLP vaccination of nonhuman primates elicited both complement-mediated lysis and ADCC (figure 1B and 1C). Although the interaction between antibodies and antigen provides the specificity of the response, the complement system is likely to provide the actual protective response by destroying antigen-coated cells. Antibodies from the eVLP-vaccinated animals were able to induce complementmediated lysis of cells expressing EBOV GP, in a dilution-dependent manner (figure 1B). ADCC caused by EBOV GP-specific antibodies coating target infected cells, making them vulnerable to killing during coculture with other cytolytic cells, also was observed when serum samples from the eVLP-vaccinated monkeys were used (figure 1C).
Cellular immune responses of eVLP-vaccinated nonhuman primates. Representative flow-cytometry data are shown for the naive control animal and for monkey VLP4, at 10 days after their third vaccination (figure 2). Although this particular eVLP-vaccinated monkey appeared to have the lowest total antibody titers, as assessed by ELISA (figure 1A), it demonstrated strong T cell responses to EBOV-peptide stimulation (figures 2 and 3). In figure 2, we plotted responses from both the naive and the eVLP-vaccinated monkeys and also show TNF-α production in T cells stimulated overnight with SEB, irrelevant peptide, or EBOV-specific peptides. The SEB-stimulated cells were used as a positive control for T cell activation and cytokine secretion, while several wells with no peptide or an irrelevant peptide (such as Lassa virus N [15, 24]) were included as negative controls, to determine the assay background (figure 2). Interestingly, specific pools fromthe majority of the eVLP-vaccinated macaques were recognized, such as GP pools 9 and 10 and NP pools 3, 4, and 11 (figure 3A and 3B). However, epitopes within the VP40 matrix protein were not strongly recognized in samples from the eVLP-vaccinated animals (figure 3C).
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Figure 2
Vigorous CD44+ T cell response to Ebola virus (EBOV) glycoprotein (GP) and nucleoprotein (NP) peptides, in nonhuman primates vaccinated with Ebola virus-like particles (eVLPs). Peripheral blood leukocytes from eVLP-vaccinated cynomolgus macaques were collected 10 days after vaccination and were used ex vivo for identification of peptides that induced intracellular tumor necrosis factor (TNF)-α in CD4+/CD44+ or CD8+/CD44+ T cells. Percentages of TNF-α-producing cells that were >2-fold higher than the background percentage (no peptide or irrelevant peptide) were considered to be positive. Representative responses to EBOV GP and NP peptides in an eVLP-vaccinated animal (VLP4) and a naive control animal are shown. SEB, staphylococcal enterotoxin B.
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Figure 3
Cellular immune responses of nonhuman primates vaccinated with Ebola virus-like particles (eVLPs) to Ebola virus (EBOV)-based peptides. Peripheral blood mononuclear cells from eVLP-vaccinated animals (VLP1-5) were collected 10 days after vaccination and were used ex vivo for identification of peptides from EBOV glycoprotein (GP; A), nucleoprotein (NP; B), and VP40 (C) that induced CD4+/CD44+ or CD8+/CD44+ T cells expressing tumor necrosis factor (TNF)-α. Percentages of TNF-α-producing cells that were >2-fold higher than the background percentage (dashed line) were considered to be positive. All cells were tested against duplicate pools of peptides, and responses to 1 set are shown here.
Protection of nonhuman primates against EBOV infection, by eVLP vaccination. All 5 eVLP-vaccinated monkeys and the single naive control monkey were challenged with ⋃1000 pfu of ZEBOV at 4 weeks after the last vaccination (figure 4A). As typically observed in cynomolgus macaques, the control monkey developed clinical and laboratory signs of EBOV infection beginning 4–5 days after challenge and died from the disease 6 days after challenge (figures 4 and 5). Results for a cohort of 23 naive control monkeys infected with the same challenge virus via the same means are shown in figure 4A, to indicate the normal course of disease and the mean time to death after EBOV challenge. The eVLP-vaccinated monkeys were completely protected against disease after lethal EBOV challenge. We observed no signs of clinical disease, such as rash, anorexia, or weight loss, in any of the eVLP-vaccinated monkeys. In addition, viremia was not detected by standard plaque assay at any of the time points when measurements were taken (figure 4B). We did observe a mild elevation (2°F–3°F) in core body temperature in 2 eVLP-vaccinated animals on day 8 after challenge, although this increase was transient and not typical of the high fever usually observed in filovirus-infected monkeys (figure 4C).
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Figure 4
Protection of nonhuman primates vaccinated with Ebola virus- like particles (eVLPs) containing glycoprotein, nucleoprotein, and VP40. Cynomolgus macaques (n=5) were vaccinated 3 times with eVLPs in RIBI adjuvant. A, Survival after challenge with 1000 pfu of Zaire Ebola virus (ZEBOV), assessed 4 weeks after the last vaccination. For the purposes of representation on the Kaplan-Meier plot, results for the control monkey in the current study were combined with those for 23 historical control animals challenged with the same seed stock of ZEBOV, at the same dose and via the same route. B, Virus titers in the plasma of ZEBOVchallenged eVLP-vaccinated monkeys were determined by use of a standard plaque assay. The laboratory values for the naive control monkey are shown from an unscheduled blood sample at 6 days after challenge, obtained immediately before euthanasia. C, Rectal temperature after challenge. Data are presented as the mean value for the eVLP-vaccinated animals and the single control animal. Error bars indicate SDs.
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Figure 5
Clinical laboratory results for nonhuman primates vaccinated with Ebola virus-like particles (eVLPs) containing glycoprotein, nucleoprotein, and VP40. Cynomolgus macaques (n=5) were vaccinated 3 times with eVLPs in RIBI adjuvant. Liver function was assessed by analysis of serum samples from eVLP-vaccinated monkeys after challenge. Levels of aspartate transaminase (AST; A), alkaline phosphatase (ALP; B), and alanine transaminase (ALT; C) were measured at the indicated time points. Platelet count (D), total white blood cell (WBC) count (E), and percentage of lymphocytes (F) in blood also were assessed after challenge and are presented as the mean (±SD) of the eVLP-vaccinated animals and the single control animal. The laboratory values for the naive control monkey are shown from an unscheduled blood sample at 6 days after challenge, obtained immediately before euthanasia.
We observed only minor hematologic and liver-enzyme changes in the eVLP-vaccinated monkeys after EBOV challenge (figure 5). Liver enzymes, such as aspartate transaminase, alkaline phosphatase, and alanine transaminase, were not altered after EBOV infection of the eVLP-vaccinated monkeys (figure 5A–5C). The numbers of circulating platelets did not decrease in the eVLP-vaccinated monkeys after EBOV challenge (figure 5D). Not surprisingly, total white blood cell (WBC) count and percentage of lymphocytes in the blood were slightly more variable in all the animals after challenge (figure 5E and 5F). As expected, the naive control animal in this experiment exhibited an increase in total WBC count, with a concomitant decrease in the percentage of circulating lymphocytes (figure 5E and 5F).
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Discussion
Numerous vaccine approaches have been used against lethal filoviral infections, including classic vaccine preparations of in activated or avirulent virus [25, 26]; viral vectors such as adenovirus, VEE, paramyxovirus, vaccinia, and vesicular stomatitis virus that encode protective filoviral proteins [24, 26–29], DNA [27, 30–32]; and other subunit vaccines [2, 26, 33]. In this study, we have shown that VLPs are a promising vaccine candidate for protection of nonhuman primates against lethal EBOV infection. Some of the advantages of VLPs as filovirus vaccines include presentation of antigen in its native form, an excellent safety profile, no interference by a vector backbone, lack of problems related to vector immunity, and induction of strong antibody and T cell responses.
In our studies, we have found high levels of antibodies in the serum of eVLP-vaccinated nonhuman primates, using 4 different assays. Not only did the vaccinated animals develop high levels of total EBOV-specific antibodies, as determined by ELISA, but they also developed antibodies that were highly active in vitro. These antibodies induced virus-neutralizing (data not shown), ADCC, and complement-mediated lysis activity (figure 1B and 1C). Data suggest that our VLP vaccines induced EBOV-specific antibodies that are multifunctional.On the basis of our studies of nonhuman primates thus far (figure 1 and authors' unpublished data), whether total levels of antibodies correlate with protection against filovirus challenge is not clear. We had shown previously that, although B cells are absolutely required for eVLP-mediated protection in mice, transfer of serum from eVLP-vaccinated mice to naive recipients did not confer protection against EBOV infection, but this result may have been dependent on the amount of antibody transferred [15]. However, the role of B cells and antibodies in protection against filoviral infection needs further investigation, and their necessity for the protection of nonhuman primates against EBOV infection is uncertain [34–38].
The eVLP-vaccinated monkeys developed strong T cell responses to EBOV epitopes, as assessed by intracellular cytokine (TNF-α) staining of peptide-stimulated PBMCs. More responses to GP and NP were observed than to the VP40 matrix protein (figure 3). Although responses to the peptides varied among the monkeys, certain epitopes were recognized by more than 1 monkey. Unfortunately, at this time, the reagents that are available to describe the T cell responses of cynomolgus macaques are limited, and further characterization of these responses may be required [39]. The importance of T cell responses in VLP-mediated protection against EBOV infection has been demonstrated by use of knockout mice: CD4+ knockout mice were protected only partially against EBOV infection, and CD8+ T cells were absolutely required [15]. On the basis of our studies of mice, induction of both EBOV-specific antibodies, to impede early viral infection and replication, and cytotoxic T cells, to destroy virus-infected cells, was found to be necessary for immunity and protection against EBOV infection. Future studies of nonhuman primates and clinical trials with humans will be required, to identify correlates of immunity for VLPs and filoviral infection.
Proper glycosylation and presentation of viral proteins, as well as vaccine dose, are critical factors for successful filovirus vaccines [14, 27, 30, 33, 40]. We previously have shown that eVLP-mediated protection against EBOV challenge is dose dependent but that the addition of adjuvant can help reduce the VLP dose and the number of injections required to mitigate protection [14–16, 18, 41]. Kinetic studies of antibody titers and T cell responses in eVLP-vaccinated nonhuman primates indicated that 3 doses of VLPs did not appear to boost the gross immune responses, compared with the responses observed after 2 vaccines (figure 1 and data not shown). Nonetheless, future studies are required in order to refine the VLP vaccination schedule, dose, and requirement for adjuvant in nonhuman primates. Studies of mice and guinea pigs have indicated that protection after only 1 VLP vaccination is an achievable goal [2, 18]. Currently, we also are examining the role of a variety of adjuvants in enhancing VLP responses.
The eVLPs provided robust protection in the vaccinated nonhuman primates. Significant clinical or laboratory signs of EBOV infection, including detectable viremia, anorexia, or considerably elevated levels of liver enzymes, were not found in the eVLP-vaccinated monkeys. However, eVLP vaccination did not induce sterile immunity against this lethal EBOV challenge. A half-log increase in total anti-EBOV antibody titers was observed in the eVLP-vaccinated monkeys at 28 days after challenge (figure 1A). Furthermore, we observed a broadening of the T cell repertoire in the eVLP-vaccinated monkeys after EBOV challenge. This was demonstrated by the recognition of T cell epitopes in proteins not included in the vaccine, such as VP24 and VP35, in lymphocytes after challenge but not before challenge (data not shown). This observation was not surprising, since we previously had noted an increase in EBOV-specific antibodies and the development of cytotoxic T lymphocytes recognizing VP24 and VP35 in eVLP-vaccinated rodents after challenge [14, 15, 18]. Since the vaccinated nonhuman primates were completely protected against clinical signs and symptoms of EBOV infection, the induction of sterile immunity by an EBOV vaccine candidate does not seem to be critical.
Although VLPs produced in mammalian cells are highly immunogenic, we currently are exploring alternative strategies for vaccine production, to accelerate the development process toward the use of eVLPs and Marburg VLPs (mVLPs) in humans. We and others have successfully generated eVLPs and mVLPs in a baculovirus expression system [13, 41]. Both insect cell-derived eVLPs and mVLPs were able to mature and activate human myeloid dendritic cells (data not shown) [41]. Furthermore, mice vaccinated with insect cell-derived eVLPs survived lethal challenge with mouse-adapted EBOV, suggesting that the baculovirus-derived eVLPs are as effective as those produced in mammalian cells [41].
Since our current data indicate that eVLPs are highly immunogenic in monkeys and stimulate virus-specific humoral and cellular responses, our next major goal is to demonstrate the efficacy of mVLPs against Marburg virus (MARV) infection [16] and also of a mixture of eVLPs and mVLPs as a panfilovirus vaccine against both EBOV and MARV infections [18]. In addition, we currently are planning preclinical experiments in preparation for future clinical trials with humans. Besides use as a vaccine, VLPs also are being used to dissect innate immune responses to filoviruses, with the goal of developing immunotherapeutics for the treatment of EBOV and MARV infections [42], as well as a safe surrogate model for the examination of filoviral replication, entry, and assembly [2].
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Acknowledgments
We especially would like to thank M. A. Fernandez, as well as D. K. Reed, S. Van Tongeren, K. Kuehl, A. Pace, N. Posten, J. Smith, C. Rice, and J. Stockman, for excellent technical assistance.
Supplement sponsorship. This article was published as part of a supplement entitled “Filoviruses: Recent Advances and Future Challenges,” sponsored by the Public Health Agency of Canada, the National Institutes of Health, the Canadian Institutes of Health Research, Cangene, CUH2A, Smith Carter, Hemisphere Engineering, Crucell, and the International Centre for Infectious Diseases.
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Footnotes
Potential conflicts of interest: K.L.W., D.L.S., M.J.A., and S.B. hold patent rights to Ebola and Marburg virus-like particle-based vaccines. G.G.O. and W.V.K.: none reported.
Financial support: Defense Threat Reduction Agency. Supplement sponsorship is detailed in the Acknowledgments.
Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the US Army.
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