SUPPLEMENT ARTICLE
Protective Efficacy of a Bivalent Recombinant Vesicular Stomatitis Virus Vaccine in the Syrian Hamster Model of Lethal Ebola Virus Infection Yoshimi Tsuda,1 David Safronetz,1 Kyle Brown,2,3 Rachel LaCasse,4 Andrea Marzi,1 Hideki Ebihara,1 and Heinz Feldmann1,3 1Laboratory
of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana; 2Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, 3Department of Medical Microbiology, University of Manitoba, Winnipeg, Canada; 4Rocky Mountain Veterinary Branch, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana
Background. Outbreaks of filoviral hemorrhagic fever occur sporadically and unpredictably across wide regions in central Africa and overlap with the occurrence of other infectious diseases of public health importance. Methods. As a proof of concept we developed a bivalent recombinant vaccine based on vesicular stomatitis virus (VSV) expressing the Zaire ebolavirus (ZEBOV) and Andes virus (ANDV) glycoproteins (VSVDG/Dual) and evaluated its protective efficacy in the common lethal Syrian hamster model. Hamsters were vaccinated with VSVDG/Dual and were lethally challenged with ZEBOV or ANDV. Time to immunity and postexposure treatment were evaluated by immunizing hamsters at different times prior to and post ZEBOV challenge. Results. A single immunization with VSVDG/Dual conferred complete and sterile protection against lethal ZEBOV and ANDV challenge. Complete protection was achieved with an immunization as close as 3 days prior to ZEBOV challenge, and 40% of the animals were even protected when treated with VSVDG/Dual one day postchallenge. In comparison to the monovalent VSV vaccine, the bivalent vaccine has slightly reduced postexposure efficacy most likely due to its restricted lymphoid organ replication. Conclusions. Bivalent VSV vectors are a feasible approach to vaccination against multiple pathogens.
Members of the genera Ebolavirus (EBOV) and Marburgvirus (MARV), family Filoviridae, are responsible for hemorrhagic fever (HF) outbreaks in humans in central Africa with high case fatality rates ranging from 40 to 90% [1–4]. The genus EBOV is divided into 5 distinct species, whereas the genus MARV contains only a single species [1]. Currently, there are no approved vaccines or effective therapeutics licensed for filovirus infections [4]. Over the past decades, multiple vaccine approaches have been developed and evaluated in animal models of EBOV and MARV including DNA vaccination, subunit
Potential conflicts of interest: none reported. Correspondence: Heinz Feldmann, MD, PhD, Rocky Mountain Laboratories, 903 South 4th St, Hamilton, MT (
[email protected]). The Journal of Infectious Diseases 2011;204:S1090–S1097 Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2011. 0022-1899 (print)/1537-6613 (online)/2011/204S3-0046$14.00 DOI: 10.1093/infdis/jir379
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vaccines, replication-incompetent and competent viral vectors, and viruslike particles [5–7]. One of the current promising vaccine approaches is based on live-attenuated recombinant vesicular stomatitis viruses (VSVs) expressing single foreign glycoproteins (GPs) as immunogens replacing the VSV glycoprotein (VSV G; monovalent vaccine vector [Figure 1A]). These VSV-based vaccines have proven potent protective efficacy in nonhuman primates (NHPs) against homologous (strains within species) challenge with highly pathogenic viruses, including EBOV, MARV, and Lassa viruses [8–14]. Cross-species protection, however, has not been achieved in NHPs with any of those monovalent VSV vaccine vectors unless vectors were used in a blended approach [5, 7, 9, 13–14]. Development of multivalent vaccine strategies is desirable to control emerging infectious diseases such as viral HF including those caused by filoviruses for several reasons: (1) Central Africa is endemic for several EBOV species and MARV; (2) filoviral HF endemicity zones
Figure 1. Generation and in vitro characterization of the recombinant vesicular stomatitis viruses (VSVs). A, Schematic diagram of recombinant VSVs. Two glycoproteins derived from Zaire ebolavirus (GP) and Andes virus (GPC) were inserted between the matrix and polymerase genes replacing the VSV G gene. B, In vitro growth kinetics. Vero cells were infected with recombinant VSVs at a multiplicity of infection of 0.0001. Supernatants were collected at the indicated time points, and virus titers were determined by measuring the 50% tissue culture infectious dose (TCID50) with standard deviation (n 5 3 independent experiments). Recombinant VSVs replicated to significantly lower levels than VSV wild type at 36 hours (* P , .1) and 48 hours (** P , .01) postinoculation (by Student t test). ANDV, Andes virus; L, RNA-dependent RNA polymerase gene; M, matrix protein gene; N, nucleoprotein gene; P, phosphoprotein gene; VSVDG, VSV lacking VSV glycoprotein (G); VSVwt, VSV wild type; VSVDG/ZEBOV, VSV expressing Zaire ebolavirus glycoprotein (GP); VSVDG/ANDV, VSV expressing Andes virus glycoprotein precursor (GPC); VSVDG/Dual, VSV expressing ZEBOV GP and ANDV GPC; ZEBOV, Zaire ebolavirus.
overlap with the endemic areas of other infectious diseases with public health impact including malaria and arbovirus infections; and (3) all filovirus species except Reston ebolavirus are considered potential biothreat agents. Therefore, multivalent vaccines may achieve a broad protection against multiple filovirus species. In addition, they could confer simultaneous protection against more prominent infectious disease problems and thus make filovirus vaccines better acceptable. More recently, the first multivalent vaccine approaches for filoviruses have been developed [15–17]. We are interested in using multivalent replication-competent VSV-based vectors expressing foreign glycoproteins replacing the VSV G (Figure 1). As a proof-of-concept study of a bivalent VSV-based vaccine we decided to generate a bivalent VSV-based vaccine expressing the ZEBOV GP and the Andes virus (ANDV) GPC. ANDV is a New World hantavirus and the major cause of
hantavirus pulmonary syndrome (HPS) in South America with high case fatality [18, 19]. Both pathogens obviously do not have overlapping endemicity zones but share a common lethal small animal disease model, the Syrian hamster (Mesocricetus auratus), which would allow objective evaluation of protective efficacy. Hamsters inoculated with mouse-adapted Zaire ebolavirus (MA-ZEBOV) develop severe illness including uncontrolled cytokine expression/release and coagulation abnormalities, hallmarks of Ebola HF in humans and NHPs, and succumb to infection within 4–7 days [20]. Hamsters infected with ANDV develop an acute respiratory distress syndrome similar to human HPS starting on days 7–9 and succumb to infection within 24–36 hours after the appearance of clinical signs [21]. Here we show that bivalent VSV vaccine vectors conferred complete and sterile protection following a single immunization against lethal challenge with both MA-ZEBOV and ANDV.
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MATERIALS AND METHODS
were collected from 3 wells per virus. Ten-fold dilutions of supernatants were inoculated onto 90% confluent monolayers of Vero cells and incubated at 37°C. The 50% end-point dilutions were expressed as 50% tissue culture infectious dose (TCID50). The values from the 3 wells per time point were averaged, and the standard error of the mean was calculated.
Cells and Viruses
Vaccine Efficacy Studies
Vero and 293T cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum. MA-ZEBOV and ANDV, strain Chile 9717869, were kindly provided by Michael Bray and Connie Schmaljohn (US Army Medical Research Institute of Infectious Diseases), respectively, and were propagated in Vero cells [22–24]. Virus infectivity titers (focus-forming units [FFUs]) for MA-ZEBOV and ANDV were obtained as described previously [25, 26] by counting the number of infected cell foci detected in an indirect immunofluorescent antibody assay using rabbit polyclonal anti-EBOV VP40 (kindly provided by Dr Y. Kawaoka, University of Wisconsin–Madison) or commercial anti-ANDV NP (AUSTRAL Biologicals) antibodies, respectively.
Syrian hamsters (male, 5–6 weeks) were purchased from Harlan Laboratories Inc. To evaluate the protective efficacy of VSVDG/ Dual, groups of hamsters were immunized with 105 PFU of either VSVDG/Dual (n 5 21), VSVDG/ZEBOV (n 5 15), or VSVDG/ANDV (n 5 9) via intraperitoneal (i.p.) injection. At 28 days postvaccination, the hamsters were challenged i.p. with 100 LD50 of either MA-ZEBOV or ANDV. On day 4 (MA-ZEBOV challenge) and days 6 and 9 (ANDV challenge) postinfection, 3 hamsters from each group were euthanized, and tissues (lung, liver, and spleen) and blood were collected for further analysis. The remaining hamsters were monitored for disease progression for 43 days postchallenge. Blood samples were collected at the end to monitor antibody responses.
Generation of Recombinant VSV Expressing ANDV GPC and ZEBOV GP
Time to Immunity Studies and Postexposure Treatment
Animals were even partially protected when treated one day after ZEBOV challenge. Overall, the bivalent VSV vaccine is as potent in prophylaxis as the monovalent vectors but may be less potent for application in postexposure treatment.
The monovalent recombinant VSV expressing ZEBOV GP (VSVDG/ZEBOV) or ANDV GPC (VSVDG/ANDV) and the bivalent recombinant VSV expressing ZEBOV GP and ANDC GPC (VSVDG/Dual) were generated as described previously using the infectious clone of VSV (pVSVXN2 plasmid, kindly provided by J. Rose, Yale University, New Haven; Figure 1A) [27–29]. In brief, the open reading frames (ORFs) encoding for the ZEBOV GP (strain Mayinga) and ANDV GPC (strain Chile 9717869) were amplified by polymerase chain reaction (PCR) from plasmids containing the appropriate complementary DNA. To generate the bivalent vector the ANDV GPC ORF was inserted into the monovalent pVSVXN2DG vector carrying the ZEBOV GP ORF using the MluI and BlnI sites (upstream of the ZEBOV GP ORF; Figure 1A). All plasmids were sequence confirmed prior to use in transfection and rescue experiments. Following rescue recombinant VSVs were propagated in Vero cells, and infectivity titers were determined by plaque assay (plaque-forming units [PFUs]). Incorporation of the ZEBOV GP and ANDV GPC into VSV particles was demonstrated by Western blot assay using antiEBOV GP (kindly provided by Dr. A. Takada, Hokkaido University, Sapporo) or commercial anti-ANDV GN and GC antibodies (AUSTRAL Biologicals), respectively (data not shown). In Vitro Growth Kinetics of Recombinant VSV Viruses
Vero cells were inoculated with VSV wild type (VSVwt), VSVDG/ZEBOV, VSVDG/ANDV, or VSVDG/Dual at a multiplicity of infection (MOI) of 0.0001. Virus was allowed to adsorb for 1 hour; then the supernatant was removed, and cells were washed twice with DMEM. At set time points, the supernatants S1092
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To determine the minimum time required for inducing protective immunity, groups of hamsters (n 5 5) were immunized once with DMEM (control), VSVDG/Dual, or VSVDG/ZEBOV (105 PFU) on day 14, 7, or 3 prior to MA-ZEBOV challenge (100 LD50). To evaluate postexposure protective vaccine efficacy, groups of hamsters (n 5 5) were challenged with MA-ZEBOV (100 LD50) and treated once with DMEM, VSVDG/Dual, or VSVDG/ZEBOV at day 0 (time of challenge) or day 1 or 2 after challenge. Animals were monitored daily for disease progression for 28 days. Vaccine Vector Replication and Tropism In Vivo
To examine the replication and tropisms of the recombinant VSV vaccines in vivo, hamsters immunized once with 105 PFU of either VSVDG/Dual or VSVDG/ZEBOV (n 5 3) were euthanized on day 1, 2, or 3 postvaccination, and tissues (lung, liver, spleen, and lymph nodes) and blood were collected for virology. Challenge Virus Titration
MA-ZEBOV and ANDV RNA were detected by quantitative real-time polymerase chain reaction (RT-PCR). For this purpose, pieces of tissue (30 mg) were placed into individual tubes containing 1 mL of RNAlater buffer (Qiagen) overnight, mechanically homogenized in 600 lL of RLT lysis buffer (Qiagen), and clarified by low-speed centrifugation. In addition, 140 lL of cardiac blood was mixed with 560 lL lysis buffer AVL (Qiagen). RNA was extracted from these samples using the RNeasy (solid tissue) or QIAamp (blood) extraction kit (Qiagen). RT-PCR was conducted on RNA extracts using a Rotor-Gene RG-3000 instrument (Corbett Life Science). Samples were quantified
against a standard curve of MA-ZEBOV or ANDV RNA extracted from 10-fold serial dilutions of viral stocks with predetermined titers. Virus titers were shown as FFU equivalent of RNA copies in 200 ng of total tissue RNA. Vaccine Virus Titration
To detect VSV replication, a piece of collected tissue was mechanically homogenized in 10-times volume of DMEM. Tenfold dilutions of homogenates were inoculated onto 80%–90% confluent monolayers of Vero cells and incubated at 37°C. The infectivity of VSV was determined as TCID50. Antibody Detection
Immunoglobulin G (IgG) antibody responses to MA-ZEBOV and ANDV were quantified by enzyme-linked immunosorbent assay (ELISA) using either purified ZEBOV GP-deltaTM, EBOV-like particles (VP40), or ANDV NP, respectively [24, 30–32]. Briefly, 96 well microtiter plates (NUNC) were coated with the antigens in phosphate-buffered saline (PBS) at 4°C overnight and subsequently were blocked with 5% skim milk in PBS containing 0.05% Tween 20 (PBST) for 2 hours at 4°C. After 3 washes with PBST, 50 lL of diluted serum samples were added, and the plates were incubated for 1 hour at 37°C. Bound antibodies were detected after 3 washes using the anti-hamster secondary antibody conjugated with horseradish peroxidase (HRP). Following incubation for 1 hour at 37°C, bound HRP was detected using the ABTSÒ Peroxidase Substrate System (KPL). The absorbance at 405 nm was determined with a microplate spectrophotometer. Statistical Analysis
Statistical analyses were performed using a 2-tailed Student t test. The Kaplan–Meier log-rank test was used to compare survival rates in the challenge study. Biosafety and Animal Work Statements
All infectious in vitro and in vivo studies were performed in Biosafety Level–4 containment at the Integrated Research Facility of the Rocky Mountain Laboratories, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Animal studies were approved by the Institutional Animal Care and Use Committee and were performed by certified staff in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care. RESULTS Construction and In Vitro Characterization of Monovalent and Bivalent VSV Vectors
Two monovalent VSV vectors expressing the ZEBOV GP (VSVDG/ZEBOV) and ANDV GPC (VSVDG/ANDV) and a single bivalent VSV vector expressing both antigens (VSVDG/ Dual) were generated using the infectious clone system for VSV as
described previously [29] (Figure 1A). Virus rescue was performed in a mixture of 293T and Vero cells and included a blind passage in Vero cells. Titers of rescued viruses ranged from 106 to 107 PFU/mL. The intracellular expression and particle incorporation of ZEBOV GP and ANDV GPC into VSV particles were demonstrated by Western blot using mouse monoclonal antibodies against the ZEBOV GP or ANDV GN and GC, respectively (data not shown). For in vitro characterization, Vero cells were infected with VSVDG/Dual, VSVDG/ZEBOV, VSVDG/ ANDV, and VSVwt (MOI 5 0.0001), and supernatants were analyzed for infectivity over a period of 96 hours. In comparison to VSVwt, which replicated faster and reached maximum titers of 5 3 109 TCID50/mL already at 36 hours, all recombinant VSVs were attenuated, and virus titers differed significantly from VSVwt at 36 (P , .1) and 48 hours (P , .01) postinfection (Figure 1B). The monovalent VSVDG/ZEBOV replicated slightly faster than the bivalent VSVDG/Dual, reaching maximum titers of close to 1 3 108 TCID50/mL at 60 or 72 hours postinfection, respectively. VSVDG/ANDV was most attenuated, reaching maximum titers at 96 hours. Interestingly, all 4 viruses reached similar titers at 84 hours postinfection. Overall, recombinant VSVs were attenuated in vitro, and the degree of attenuation did not correlate with the number of foreign gene insertions. Bivalent VSV Vaccine Protects Hamsters Against Lethal Ebola or Andes Virus Challenge
For the initial experiment, we examined whether VSVDG/Dual can elicit protective immunity against both ZEBOV and ANDV infections. Hamsters were immunized with VSVDG/Dual (n 5 12), VSVDG/ZEBOV (n 5 6), or VSVDG/ANDV (n 5 6) followed by lethal challenge of half of the animals with MA-ZEBOV or ANDV 28 days after vaccination. Animals immunized with VSVDG/ANDV and challenged with MA-ZEBOV or immunized with VSVDG/ZEBOV and challenged with ANDV succumbed to infection within 6 or 9 days, respectively (Table 1). Hamsters immunized with VSVDG/ANDV or VSVDG/ZEBOV and challenged with ANDV and MA-ZEBOV, respectively, were completely protected against disease. All animals vaccinated with VSVDG/Dual were completely protected against lethal challenge with either virus, MA-ZEBOV or ANDV. The anti-ZEBOV GP antibody responses in animals immunized with the different VSV vaccines were tested using an IgG ELISA. All hamsters immunized with VSVDG/Dual and VSVDG/ZEBOV had developed high titers (titers ranged from 25,600 to 51,200) of antiZEBOV GP antibodies 28 days after immunization (Table 1). These titers did not increase or only marginally increased after challenge, indicating induction of strong humoral immune responses to vaccination and little or no challenge virus replication. To further investigate challenge virus replication we examined antibody responses to challenge virus–specific antigens not expressed by the VSV vaccines (ZEBOV VP40 and ANDV NP). With the exception of 2 hamsters in the VSVDG/
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Table 1. Protective Efficacy of Bivalent Recombinant Vesicular Stomatitis Virus Anti-ZEBOV GP
Anti-ZEBOV VP40
Anti-ANDV NP
Vaccine virus
Challenge virus
Pre
Post
Pre
Post
Pre
Post
VSVDG/Dual
MA-ZEBOV
25 600
25 600
,100
,100
,100
,100
Survival/total 6/6
VSVDG/Dual
ANDV
25 600
38 400
,100
,100
,100
,100
6/6
VSVDG/ZEBOV
MA-ZEBOV
25 600
51 200
,100
,100
,100
,100
3/3
VSVDG/ZEBOV
ANDV
25 600
ND
,100
ND
,100
ND
0/3
VSVDG/ANDV
MA-ZEBOV
,100
ND
,100
ND
,100
ND
0/3
VSVDG/ANDV
ANDV
,100
,100
,100
,100
,100
,100
3/3
NOTE. Serum samples were collected 1 day before challenge (pre) or 43 days after challenge (post). Immunoglobulin G end-point dilution titers are presented as average titers of all animals in each group (n 5 3 or 6). ANDV, Andes virus; MA-ZEBOV, mouse-adapted ZEBOV; ND, not determined due to prior animal loss; VSVDG, vesicular stomatitis virus (VSV) lacking VSV glycoprotein (G); VSVDG/ANDV, VSV expressing ANDV glycoprotein precursor (GPC); VSVDG/Dual, VSV expressing ZEBOV GP and ANDV GPC; VSVDG/ZEBOV, VSV expressing ZEBOV glycoprotein (GP); ZEBOV, Zaire ebolavirus.
ANDV-immunized group with low IgG titers to ANDV NP (1:100), no antibody responses (,1:100) were detected in surviving animals, supporting lack of challenge virus replication (Table 1). These results suggest that all 3 VSV vaccine vectors confer complete and largely sterile protection after a single immunization against lethal MA-ZEBOV and ANDV challenge. Detection of Challenge Virus Replication Following Immunization
To confirm sterile immunity in vaccinated animals after challenge with ZEBOV and ANDV, groups of 3 hamsters were sacrificed at day 4 (MA-ZEBOV) and day 6 and 9 postinfection
(ANDV). Blood, lung, liver, and spleen were collected from each animal, and samples were analyzed for the presence of viral RNA by RT-PCR. With the exception of one animal on day 9 post– ANDV challenge (spleen), all other animals immunized with VSVDG/Dual showed no detectable levels of MA-ZEBOV or ANDV viral RNA in any of the tissues and blood (Figure 2). RNA levels detected in the spleen of the above-mentioned animal were extremely low (,101 FFU equivalent/200 ng RNA), making virus isolation unlikely from this specimen. In contrast, significant higher viral RNA levels (between 102 and 106 FFU equivalent/200 ng RNA) were detected in control animals from groups that did not survive challenge. These data confirm the
Figure 2. Protective efficacy of recombinant VSVs against lethal challenge with mouse-adapted (MA) ZEBOV or ANDV. A, Hamsters were vaccinated intraperitoneally (i.p.) with a single dose (105 plaque-forming units [PFU]) of VSVDG/Dual, VSVDG/ZEBOV, and VSVDG/ANDV and were challenged with MA-ZEBOV (100 LD50) 28 days after immunization. Four days after challenge, 3 hamsters of each group were euthanized to determine viral load in blood and certain tissues using a real-time polymerase chain reaction (RT-PCR) assay. B, Hamsters were vaccinated i.p. with a single dose (105 PFU) of VSVDG/ Dual or VSVDG/ZEBOV and were challenged with ANDV (100 LD50). Six and 9 days after challenge, 3 hamsters of each group were euthanized to determine viral load in blood and certain tissues using an RT-PCR assay. Presence of viral RNA is shown here as focus-forming unit (FFU) equivalents calculated from virus RNA copies in 200 ng of total RNA. Standard deviation was calculated from 3 animals. VSVDG, VSV lacking VSV glycoprotein (G); VSVDG/ZEBOV, VSV expressing ZEBOV glycoprotein (GP); VSVDG/ANDV, VSV expressing ANDV glycoprotein precursor (GPC); VSVDG/Dual, VSV expressing ZEBOV GP and ANDV GPC. S1094
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Figure 3. Survival curves of vaccinated (preexposure) and treated (postexposure) hamsters. Groups of 5 hamsters were immunized i.p. with a single dose (105 PFU;) of VSVDG/Dual (black), VSVDG/ZEBOV (gray), or Dulbecco's Modified Eagle's Medium (white). Immunization occurred either (A ) prior to (days –14, –7, and –3) or (B) after (days 0, 11, and 12) i.p. challenge with 100 LD50 of MA-ZEBOV. VSVDG/Dual showed a significant reduction of survival rate on day 1 (P , .1) and day 2 (P , .01) posttreatment (by Kaplan–Meier log-rank test) compared with VSVDG/ZEBOV. The symbols relate to days of immunization/treatment performed prior to (–) or post (1) infection.
serology data and support the notion that all 3 vaccines confer sterile immunity in vaccinated animals. Time to Immunity and Postexposure Protection
Single immunization with VSVDG/Dual 28 days before challenge was able to completely protect hamsters from lethal MA-ZEBOV challenge. To determine the minimum time required for the induction of sufficient protective immunity, groups of 5 hamsters were immunized once with VSVDG/Dual (105 PFU), VSVDG/ZEBOV (105 PFU), or DMEM (control) 14, 7, and 3 days prior to MA-ZEBOV challenge. All VSVDG/Dualand VSVDG/ZEBOV-vaccinated animals survived without showing clinical signs (Figure 3A, overlapping black and gray lines). In contrast, control animals inoculated with DMEM succumbed to disease within 6 days after challenge (Figure 3A, white lines). The results indicate a short time to immunity for both the monovalent and bivalent vaccine vectors. To evaluate the potential for postexposure vaccination, hamsters were inoculated with a lethal dose of MA-ZEBOV and treated with VSVDG/Dual (105 PFU), VSVDG/ZEBOV (105 PFU), or DMEM (n 5 5) on day 0 (with challenge) or day 1 or 2 postchallenge. As shown in Figure 3B, animals treated with VSVDG/Dual (Figure 3, black lines) were not fully protected, with 80% (4 of 5 animals; P 5 .3), 40% (2 of 5 animals; P 5 .05), and 0% (0 of 5 animals; P , .01) survival when treated 0, 1, or 2 days after infection, respectively. In contrast, all animals treated with VSVDG/ZEBOV (Figure 3B, gray lines) on days 0 and 1 postchallenge survived, but the animals treated on day 2 postchallenge died between days 7 and 15 after challenge. All DMEM-treated animals succumbed to infection within 6 days postchallenge (Figure 3B, white line). These data indicate a reduced potential for postexposure treatment for the bivalent vector compared with the monovalent one.
Vaccine Vector Replication Associated With Immunization
In order to determine factors for the difference in postexposure efficacy, replication and tissue tropisms of VSVDG/Dual and VSVDG/ZEBOV in hamsters were compared. Groups of 3 hamsters were euthanized each day between days 1 and 3 following vaccination with VSVDG/Dual (105 PFU) or VSVDG/ ZEBOV (105 PFU). Blood, lung, liver, spleen, and certain lymph node (mesenteric, inguinal, cervical, and axillary) samples were collected from each animal and analyzed for infectivity using a TCID50 assay. The replication of VSVDG/Dual was restricted to lymphoid tissues, with very low levels of viremia in one animal (Figure 4A). In contrast, replication of VSVDG/ ZEBOV was already detected on day 1 postimmunization in blood and all tissues analyzed (Figure 4B). Compared with VSVDG/ZEBOV, which showed the highest titers already at day 1 postimmunization, VSVDG/Dual titers were low and gradually increased over time, indicating further attenuation of this vector expressing 2 foreign genes. Furthermore, we detected the ZEBOV GP and ANDV GPC genes in tissues collected at day 3 postvaccination, indicating that both genes are stable in vitro and expressed (data not shown). DISCUSSION In this study, a bivalent recombinant VSV vaccine expressing 2 different immunogens derived from ZEBOV (GP) and ANDV (GPC) resulted in complete and sterile protection of hamsters upon single immunization against lethal challenge with MA-ZEBOV or ANDV. Compared with the monovalent vaccine vector expressing ZEBOV GP, VSVDG/Dual conferred similar pre-exposure protection but showed slightly reduced postexposure efficacy. Thus, VSV vectors provide an effective approach for bivalent vaccine strategies.
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Figure 4. Vaccine vector propagation following immunization. On day 1, 2, or 3 after immunization with a single dose (105 PFU) of (A) VSVDG/Dual or (B ) VSVDG/ZEBOV, groups of 3 hamsters were euthanized, and blood and tissue samples were collected for viremia levels and tissue titers. The following organs were analyzed: lung, liver, spleen, mesenteric lymph node (MLN), inguinal lymph node (ILN), cervical lymph node (CLN), and axillary lymph node (ALN). Virus titers are shown as TCID50 with standard deviation (n 5 3). Student t test was performed between VSVDG/Dual and VSVDG/ZEBOV. * P , .05; ** P , .01.
EBOV and ANDV belong to different families of viruses and show distinct biological characteristics in their viral life cycle and pathogenesis. ZEBOV infection is characterized by its rapid onset and high case fatality rates of up to 90% [2, 3]. The disease is caused by systemic viral replication, immunosuppression, abnormal systemic inflammatory response, vascular leakage, and coagulation abnormalities resulting in multiorgan failure [4, 33–35]. On the other hand, the clinically affected organ of ANDV-induced HPS is the lung, and ANDV infection presents initially with influenza-like symptoms followed by severe pulmonary edema resulting in an acute respiratory distress syndrome with a case fatality rate as high as 40% [19, 36]. In the hamster model, MA-ZEBOV replicates systemically affecting multiple organs leading to coagulation abnormality in the terminal stage [20] and death within 4–6 days. Following challenge with ANDV, infected hamsters appear normal for almost 7–8 days, at which time they quickly develop clinical signs of an acute respiratory distress, HPS, followed by death within 24–36 hours [21, 24]. The differences in virus biology, clinical disease, and host response associated with the 2 infections underscore the potency of the new recombinant bivalent VSV vaccine described here in inducing simultaneous protection after a single immunization. This offers promising opportunities for the development of cross-protective vaccines against cocirculating pathogens with similar or distinct biology and pathogenicity. Even more notable is the still impressive but slightly reduced postexposure efficacy of VSVDG/Dual against lethal challenge with MA-ZEBOV. Growth comparison in vitro and in vivo clearly demonstrated further attenuation of this recombinant vaccine vector compared with the monovalent VSVDG/ZEBOV (Figures 1B and 4). Interestingly, VSVDG/Dual replicated mainly in lymphoid organs and virus titers were lower compared S1096
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with VSVDG/ZEBOV. Conversely, VSVDG/ZEBOV replicated well in all tissues analyzed, and displayed high-level viremia on days 1 and 2 postvaccination (Figure 4). Mild temporary viremia was also detected in NHPs after monovalent VSV vaccination [9, 12]. In future the impact of multiple gene insertions into the VSV genome on virus replication needs to be determined. From our results it certainly seems that the replication competency of the vectors is a major factor for the protective efficacy, especially for postexposure treatment. From this proof-of-concept study we can conclude that multivalent VSV vaccine vectors are promising prophylactic approaches to address the cocirculation of multiple filovirus species in a defined geographic zone. They also might provide simultaneous protection against cocirculating distinct pathogens such as Lassa virus or important arbovirus infections. The model here was chosen because of the common well-defined lethal hamster model for ZEBOV and ANDV to study protective efficacy, but efficacy testing in NHPs needs to be done to confirm the concept. In the short term we will generate multivalent VSV-based vaccine vectors for rare emerging pathogens with overlapping endemic areas such as filoviruses, arenaviruses, and certain bunyaviruses. In the long term we will focus on multivalent vaccine approaches that combine rare emerging pathogens, such as filoviruses, with important regional infectious disease problems, such as malaria or arboviral infections. Success here might also help to gain better acceptance for filovirus vaccines, which are still controversially discussed in regard to their utility and applicability. Funding This work was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.
Acknowledgments We thank the members of Rocky Mountain Veterinary Branch (Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health [DIR, NIAID, NIH]), in particular Sandra Skorupa and Kathleen Meuchel, for assistance with animal care, and Friederike Feldmann, Office of Research Operation (DIR, NIAID, NIH), for assistance in high containment training and operation.
18. 19. 20.
References
21.
1. Feldmann H, Geisbert TW, Jahrling PB, et al. Virus taxonomy: Eighth report of the International Committee on Taxonomy of Viruses. In: Fauquet C, Mayo MA, Maniloff J, Desselberger U, Ball LA eds: Filoviridae. London: Elsevier/Academic Press, 2004; 645–53. 2. Pourrut X, Kumulungui B, Wittmann T, et al. The natural history of Ebola virus in Africa. Microbes Infect 2005; 7:1005–14. 3. Sanchez A, Geisbert T, Feldmann H. Filoviridae: Marburg and Ebola viruses. In: knipe D, Howley P, eds: Fields virology. 5th ed. Philadelphia: Lippincott Williams & Wilkins, 2006; 1409–48. 4. Feldmann H, Geisbert TW. Ebola haemorrhagic fever. Lancet 2011; 377:849–62. 5. Geisbert TW, Bausch DG, Feldmann H. Prospects for immunisation against Marburg and Ebola viruses. Rev Med Virol 2010; 20:344–57. 6. Richardson JS, Dekker JD, Croyle MA, Kobinger GP. Recent advances in Ebolavirus vaccine development. Hum Vaccin 2010; 6:439–49. 7. Falzarano D, Geisbert TW, Feldmann H. Progress in filovirus vaccine development—evaluating the potential for clinical use. Expert Rev Vaccines 2011; 10:63–77. 8. Geisbert TW, Jones S, Fritz EA, et al. Development of a new vaccine for the prevention of Lassa fever. PLoS Med 2005; 2:e183. 9. Jones SM, Feldmann H, Stroher U, et al. Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses. Nat Med 2005; 11:786–90. 10. Daddario-DiCaprio KM, Geisbert TW, Geisbert JB, et al. Crossprotection against Marburg virus strains by using a live, attenuated recombinant vaccine. J Virol 2006; 80:9659–66. 11. Feldmann H, Jones SM, Daddario-DiCaprio KM, et al. Effective postexposure treatment of Ebola infection. PLoS Pathog 2007; 3:e2. 12. Geisbert TW, Daddario-Dicaprio KM, Lewis MG, et al. Vesicular stomatitis virus-based Ebola vaccine is well-tolerated and protects immunocompromised nonhuman primates. PLoS Pathog 2008; 4:e1000225. 13. Geisbert TW, Geisbert JB, Leung A, et al. Single-injection vaccine protects nonhuman primates against infection with Marburg virus and three species of Ebola virus. J Virol 2009; 83:7296–304. 14. Geisbert TW, Feldmann H. Vesicular stomatitis virus-based vaccines against Ebola and Marburg virus infections. J Infect Dis 2011. doi: 10.1093/infdis/jir349. 15. Pushko P, Geisbert J, Parker M, Jahrling P, Smith J. Individual and bivalent vaccines based on alphavirus replicons protect guinea pigs against infection with Lassa and Ebola viruses. J Virol 2001; 75:11677–85. 16. Wang D, Raja NU, Trubey CM, et al. Development of a cAdVax-based bivalent Ebola virus vaccine that induces immune responses against both the Sudan and Zaire species of Ebola virus. J Virol 2006; 80:2738–46. 17. Pratt WD, Wang D, Nichols DK, et al. Protection of nonhuman primates against two species of Ebola virus infection with a single
22.
23. 24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34. 35.
36.
complex adenovirus vector. Clin Vaccine Immunol 2010; 17:572–81. Enria DA, Briggiler AM, Pini N, Levis S. Clinical manifestations of New World hantaviruses. Curr Top Microbiol Immunol 2001; 256:117–34. Khan AS, Young JC. Hantavirus pulmonary syndrome: at the crossroads. Curr Opin Infect Dis 2001; 14:205–9. Ebihara H, Kawaoka Y, Feldmann H. Pathogenesis of filoviruses in small animal models [abstract 2S-7]. In: Program and abstracts of the 5th International Symposium of Filoviruses. Tokyo, 2010;29 pp. Hooper JW, Larsen T, Custer DM, Schmaljohn CS. A lethal disease model for hantavirus pulmonary syndrome. Virology 2001; 289:6–14. Bray M, Davis K, Geisbert T, Schmaljohn C, Huggins J. A mouse model for evaluation of prophylaxis and therapy of Ebola hemorrhagic fever. J Infect Dis 1998; 178:651–61. Ebihara H, Takada A, Kobasa D, et al. Molecular determinants of Ebola virus virulence in mice. PLoS Pathog 2006; 2:e73. Safronetz D, Hegde NR, Ebihara H, et al. Adenovirus vectors expressing hantavirus proteins protect hamsters against lethal challenge with Andes virus. J Virol 2009; 83:7285–95. Ksiazek TG, Rollin PE, Williams AJ, et al. Clinical virology of Ebola hemorrhagic fever (EHF): virus, virus antigen, and IgG and IgM antibody findings among EHF patients in Kikwit, Democratic Republic of the Congo, 1995. J Infect Dis 1999; 179(Suppl 1):S177–87. Ebihara H, Yoshimatsu K, Ogino M, et al. Pathogenicity of Hantaan virus in newborn mice: genetic reassortant study demonstrating that a single amino acid change in glycoprotein G1 is related to virulence. J Virol 2000; 74:9245–55. Schnell MJ, Buonocore L, Kretzschmar E, Johnson E, Rose JK. Foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles. Proc Natl Acad Sci U S A 1996; 93:11359–65. Takada A, Feldmann H, Stroeher U, et al. Identification of protective epitopes on Ebola virus glycoprotein at the single amino acid level by using recombinant vesicular stomatitis viruses. J Virol 2003; 77:1069–74. Garbutt M, Liebscher R, Wahl-Jensen V, et al. Properties of replicationcompetent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses. J Virol 2004; 78:5458–65. Sullivan NJ, Geisbert TW, Geisbert JB, et al. Accelerated vaccination for Ebola virus haemorrhagic fever in non-human primates. Nature 2003; 424:681–4. Wahl-Jensen V, Kurz SK, Hazelton PR, et al. Role of Ebola virus secreted glycoproteins and virus-like particles in activation of human macrophages. J Virol 2005; 79:2413–9. Nakayama E, Yokoyama A, Miyamoto H, et al. Enzyme-linked immunosorbent assay for detection of filovirus species-specific antibodies. Clin Vaccine Immunol 2010; 17:1723–8. Geisbert TW, Hensley LE. Ebola virus: new insights into disease aetiopathology and possible therapeutic interventions. Expert Rev Mol Med 2004; 6:1–24. Mahanty S, Bray M. Pathogenesis of filoviral haemorrhagic fevers. Lancet Infect Dis 2004; 4:487–98. Geisbert TW, Young HA, Jahrling PB, et al. Pathogenesis of Ebola hemorrhagic fever in primate models: evidence that hemorrhage is not a direct effect of virus-induced cytolysis of endothelial cells. Am J Pathol 2003; 163:2371–82. Simpson SQ, Spikes L, Patel S, Faruqi I. Hantavirus pulmonary syndrome. Infect Dis Clin North Am 2010; 24:159–73.
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