Single dose attenuated Vesiculovax vaccines protect primates against Ebola Makona virus

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Abstract

The family Filoviridae contains three genera, Ebolavirus (EBOV), Marburg virus, and Cuevavirus ¹. Some members of the EBOV genus, including Zaire ebolavirus (ZEBOV), can cause lethal hemorrhagic fever in humans. During 2014 an unprecedented ZEBOV outbreak occurred in West Africa and is still ongoing, resulting in nearly 10,000 deaths, and causing global concern of uncontrolled disease. To meet this challenge a rapid acting vaccine is needed. Many vaccine approaches have shown promise in being able to protect nonhuman primate (NHPs) against ZEBOV ². In response to the current ZEBOV outbreak several of these vaccines have been fast tracked for human use. However, it is not known whether any of these vaccines can provide protection against the new outbreak Makona strain of ZEBOV. One of these approaches is a first generation recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing the ZEBOV glycoprotein (GP) (rVSV/ZEBOV). To address safety concerns associated with this vector, we developed two candidate, further attenuated rVSV/ZEBOV vaccines. Both attenuated vaccines produced an approximately ten-fold lower vaccine-associated viremia compared to the first generation vaccine and both provided complete, single dose protection of macaques from lethal challenge with the Makona outbreak strain of ZEBOV.

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Effective Post-Exposure Treatment of Ebola Infection

Heinz Feldmann, Steven M. Jones, Kathleen M Daddario-DiCaprio … (2007)

Introduction Editor's Note: The potential efficacy of pre- and post-exposure prophylaxis against Ebola virus infection, as well as the fundamentally important question of whether neutralizing antibodies are important for Ebola virus resistance, is addressed by a related manuscript in this issue of PLoS Pathogens. Please see doi:10.1371/journal.ppat.0030009 by Oswald et al. Infection with the filoviruses, in particular Zaire ebolavirus (ZEBOV), Sudan ebolavirus, or Marburg virus (MARV), causes a severe haemorrhagic fever (HF) in humans and nonhuman primates that is often fatal [1–3]. In addition to the sporadic outbreaks that have occurred in humans in Central Africa since 1976 and caused more than 1,800 human infections with a lethality rate ranging from 53% to 90%, Ebola virus (EBOV) has also decimated populations of wild apes in this same region [4]. At this time, there is no preventive vaccine or post-exposure treatment option available for human use. Much remains to be learned about these highly virulent viruses; however, important advances have been made over the last decade in understanding how filoviruses cause disease and in developing preventive vaccines that are protective in nonhuman primates [1,5]. For example, a recombinant replication-defective adenovirus vaccine completely protected nonhuman primates from uniformly lethal ZEBOV infection [6,7]. More recently, we generated live-attenuated recombinant vesicular stomatitis viruses (VSV) expressing the transmembrane glycoproteins (GP) of ZEBOV (VSVΔG/ZEBOVGP) and MARV (VSVΔG/MARVGP) and the glycoprotein precursor of Lassa virus (VSVΔG/LASVGPC) [8] and showed that these completely protected cynomolgus macaques against lethal challenge with the corresponding filoviruses and arenavirus [9,10]. Progress in developing therapeutic interventions against the filoviruses has been much slower [5]. Limited success was achieved in using an anticoagulant to treat EBOV infections [11], and very recently the VSV-based MARV vaccine platform (VSVΔG/MARVGP) demonstrated astonishing efficacy in post-exposure treatment of MARV-infected macaques [12]. Other than that, no post-exposure modality has been able to protect nonhuman primates against lethal filovirus infections [5,13,14]. There is clearly an urgent need to develop filovirus-specific effective post-exposure strategies to respond to future outbreaks in Central Africa, to counter acts of bioterrorism, and to treat laboratory exposures such as the recent EBOV exposures that occurred in the United States and Russian laboratories [15,16]. Post-exposure vaccine treatment is successful in preventing or modifying viral diseases such as rabies [17,18], hepatitis B [19], and smallpox [20,21] in humans, as well as MARV HF in nonhuman primates [12]. However, the faster disease course and higher lethality of ZEBOV in human and nonhuman primates may limit the success of a similar approach for EBOV HF. Here, we show remarkable efficacy of the VSV-based EBOV vaccine platform in the post-exposure treatment of rodents and nonhuman primates infected with ZEBOV. Currently, this is the most promising post-exposure treatment strategy for EBOV HF and is particularly suited for use in accidentally exposed individuals and in the control of transmission in the event of natural or deliberate outbreaks. Methods Vaccine Vectors and ZEBOV Challenge Viruses The recombinant VSV expressing the GPs of ZEBOV (strain Mayinga), MARV (strain Musoke), or Lassa virus (strain Josiah) were generated as described recently using the infectious clone for the VSV, Indiana serotype (kindly provided by J. Rose) [8]. Briefly, the appropriate open reading frames for the GPs (ZEBOV, Mayinga, MARV, Musoke) were generated by PCR, cloned into the VSV genomic vectors lacking the VSV G gene, sequenced, and originally rescued using the method described earlier [8,22]. ZEBOV (strain Kikwit) was isolated from a patient of the EBOV outbreak in Kikwit in 1995 [23]. The mouse- and guinea pig-adapted ZEBOV strains (MA-ZEBOV and GA-ZEBOV, respectively) were generated by serial passages in the different rodent species until uniformly lethal [24,25]. Animal Studies Rodents. Female BALB/c mice, 5–6 wk old, were purchased from Charles Rivers (Quebec, Canada). The animals (groups of five) were treated by intraperitoneal (i.p.) inoculation of 2 ×105 plaque forming units (pfu) of VSVΔG/ZEBOVGP into the left and right site of the abdomen (100 μl each). Naïve control animals were immunized with the same volume of Dulbecco's Mimimal Essential Medium (DMEM) by the same route. The mice were challenged i.p. with 1,000 LD50 of MA-ZEBOV into the left and right site of the abdomen (100 μl each). Female guinea pigs (Hartley strain), approximately 250 g, were purchased from Charles Rivers (Quebec, Canada). The animals (groups of six) were i.p.-treated with 2 × 105 pfu of VSVΔG/ZEBOVGP into the left and right site of the abdomen (500 μl each). Naïve control animals were immunized with the same volume of DMEM by the same route. The guinea pigs were challenged i.p. with 1,000 LD50 of GA-ZEBOV into the left and right site of the abdomen (500 μl each). All rodents (mice and guinea pigs) were weighed daily for a minimum of 11 d following challenge and observed for clinical symptoms according to an approved scoring sheet (ruffled fur, slowing activity, loss of body conditions, labored breathing, hunched posture, bleeding, paralysis). Surviving animals were kept three times longer than the death of the last control animal. All rodent work was performed in the Biosafety Level (BSL)-4 biocontainment facility at the National Microbiology Laboratory of the Public Health Agency of Canada and was approved by the Canadian Science Centre for Human and Health Animal Care Committee following the guidelines of the Canadian Council on Animal Care. Rhesus macaques. Ten healthy adult Macaca mulatta of Chinese origin (3–6 kg) were used for this study. Briefly, all ten macaques were challenged by intramuscular (i.m.) inoculation with 1,000 pfu of ZEBOV, strain Kikwit. Approximately 20–30 min after ZEBOV challenge, eight of the animals received an i.m. injection with a dose of 2 × 107 pfu of the VSVΔG/ZEBOVGP vector expressing the ZEBOV GP that was divided among four different anatomical locations (right and left triceps and right and left caudal thigh). Two animals served as experimental controls, of which one received an equivalent dose of the VSVΔG/MARVGP vector expressing the MARV GP and the other the VSVΔG/LASVGPC vector expressing the Lassa virus glycoprotein precursor by the same routes. All animals were checked twice daily for clinical symptoms of ZEBOV HF using an established score sheet. Swab samples (oral, nasal, rectal) and blood were taken prior to ZEBOV challenge and on days 3, 6, and 10 post ZEBOV challenge. Survivors were kept for more than 50 d. All nonhuman primate studies were performed in BSL-4 biocontainment at United States Army Medical Research Institute of Infectious Diseases (USAMRIID) and were approved by the USAMRIID Laboratory Animal Care and Use Committee. Animal research was conducted in compliance with the Animal Welfare Act and other Federal statues and regulations relating to animals; experiments involving animals adhere to the principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. The facility used is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Haematology and Serum Biochemistry Total white blood cell counts, lymphocyte counts, red blood cell counts, platelet counts, haematocrit values, total haemoglobin, mean cell volume, mean corpuscular volume, and mean corpuscular haemoglobin concentration were determined from nonhuman primate blood samples collected in tubes containing EDTA, by using a laser-based haematology analyzer (Beckman Coulter, http://www.beckmancoulter.com). The white blood cell differentials were performed manually on Wright-stained blood smears. Virus Detection RNA was isolated from nonhuman primate whole blood and swabs using appropriate RNA isolation kits (Qiagen, http://www1.qiagen.com). ZEBOV RNA was detected using primer pairs targeting the L genes [ZEBOV: RT-PCR, nt position 13344–13622; nested PCR, nt position 13397–13590]. The sensitivity of the ZEBOV-specific RT-PCR is approximately 0.1 pfu/ml. ZEBOV titration was performed by plaque assay on Vero E6 cells from all blood and selected organ (adrenal, ovary, lymph nodes, liver, spleen, pancreas, lung, heart, brain) and swab samples [23]. Briefly, increasing 10-fold dilutions of the samples were adsorbed to Vero E6 monolayers in duplicate wells (0.2 ml per well); thus, the limit for detection was 25 pfu/ml. Immune Responses IgG and IgM antibodies against ZEBOV were detected with an enzyme-linked immunosorbent assay (ELISA) using purified virus particles as an antigen source [6]. Neutralization assays were performed by measuring plaque reduction in a constant virus:serum dilution format as previously described [9,26]. Briefly, a standard amount of ZEBOV (∼100 pfu) was incubated with serial 2-fold dilutions of the serum sample for 60 min. The mixture was used to inoculate Vero E6 cells for 60 min. Cells were overlayed with an agar medium, incubated for 8 d, and plaques were counted 48 h after neutral red staining. End point titres were determined by the dilution of serum, which neutralized 50% of the plaques (PRNT50). Cellular Immune Responses Peripheral blood mononuclear cells were isolated from rhesus macaque whole blood samples by separation over a Ficoll gradient. Approximately 1 × 106 cells were stained for cell surface markers, granzyme B, and viral antigen using monoclonal antibodies. Staining procedures were performed as previously described [27]. Results To test the concept that the VSVΔG/ZEBOVGP vaccine may have utility as a post-exposure treatment for EBOV HF, we investigated its efficacy in two rodent models, mouse [25] and guinea pig [24], and a rhesus macaque model [11]. Initially, we treated groups of five BALB/c mice with i.p. injections of 2 × 105 pfu of the VSVΔG/ZEBOVGP vaccine 24 h prior to challenge or 30 min or 24 h post i.p. challenge with a 1,000 LD50 of the mouse-adapted ZEBOV (MA-ZEBOV) [25]. The immunization dose chosen was relatively high considering that as little as 2 × 100 pfu still conferred complete protection against the same challenge dose (unpublished data). Animals were weighed every day and scored for clinical symptoms (see Methods). Untreated control animals (naïve controls) rapidly lost weight, developed severe clinical symptoms, and died on day 6 post-challenge (Figures 1A and S1A). Surprisingly, all treated mice survived independent of the time of treatment (Figure 1A). Those animals treated 24 h prior to challenge did not show any clinical symptoms, whereas animals treated post-challenge developed mild clinical symptoms. With all protected groups, mild weight loss was observed during the first day post-challenge (Figure S1) indicating virus replication prior to clearance and survival. Figure 1 Kaplan-Meier Survival Curves for Mice and Guinea Pigs Given Post-Exposure Treatment for ZEBOV Infection (A) Mice (groups of five animals) were infected with 1,000 LD50 of MA-ZEBOV by i.p. injection. At various times points 24 h prior to challenge (○), 30 min after challenge (♦), or 24 h after challenge (▵) they were treated with 2 × 105 pfu of VSVΔG/ZEBOVGP by i.p. injection. The controls (▪) were left untreated and all died. All treated animals survived the challenge. (B) Guinea pigs (groups of six animals) were infected with 1,000 LD50 of GA-ZEBOV by i.p. injection. At various times points 24 h prior to challenge (○), 1 h after challenge (♦), or 24 h after challenge (▵) they were treated with 2 × 105 pfu of VSVΔG/ZEBOVGP by i.p. injection. The controls (▪) were left untreated and all died. Next, we treated three groups of guinea pigs (Hartley strain; six animals per group) with i.p. injection of 2 ×105 pfu of the VSVΔG/ZEBOVGP either 24 h before challenge or 1 or 24 h after challenge with 1,000 LD50 of the guinea pig-adapted ZEBOV (GA-ZEBOV) [24]. Disease progression was followed and measured as described for the mice. Untreated guinea pigs (naïve controls) showed weight loss at day 5 post-challenge progressing to death on days 7 to 9 (Figures 1B and S1B). Unlike the mice, the treatment groups were not fully protected (Figures 1 and S1). Two animals (33%) died from the group treated 24 h prior to challenge; one (17%) and three (50%) animals died from the groups treated 1 and 24 h post-challenge, respectively (Figures 1B and S1B). In all cases, the development of clinical symptoms, weight loss and time to death, were significantly delayed. All surviving animals lost weight and became sick with a degree of severity that correlated very well with disease outcome. The final survival rates were 66% for the pre-treatment group (24 h prior to challenge) and 83% and 50% in the 1- and 24-h post-treatment groups, respectively (Figures 1B and S1B). Encouraged by the success in the rodent models, we treated eight rhesus monkeys (subjects 1 to 8) with i.m. injections of the VSVΔG/ZEBOVGP vaccine (2 × 107 pfu), and two rhesus monkeys (subjects c1 and c2) with VSV control vaccines (2 × 107 pfu) (see Methods) 20 to 30 min after challenge with 1,000 pfu of ZEBOV. The immunization and challenge doses were equivalent to what had been used in previous successful pre-exposure vaccine studies [6,9]. All animals became febrile by day 6 and haematology data indicated evidence of illness by day 6, usually manifested as lymphopenia, in most of these animals (Table 1). Surprisingly, 50% of the VSVΔG/ZEBOVGP-treated animals (subjects 1, 2, 5, and 7) survived the lethal ZEBOV challenge (Figure 2A; Table 1) without showing signs of severe disease, while three VSVΔG/ZEBOVGP-treated macaques (subjects 3, 4, and 8) developed characteristic ZEBOV HF including fever, perturbations in clinical chemistry values, and macular rashes (Figure S2); these animals died on days 9 (subject 3) and 10 (subjects 4 and 8) (Figure 2A; Table 1). Notably, all VSVΔG/ZEBOVGP-treated animals that succumbed to the ZEBOV challenge (subjects 3, 4, and 8) developed plasma viraemia on day 6 between 1 × 104 and 1 × 106 pfu/ml, whereas plasma viraemia was transient in the animals that survived (subjects 1, 2, 5, and 7) and did not exceed 1 × 102 pfu/ml on day 6 (Figure 2B). The final VSVΔG/ZEBOVGP-treated macaque (subject 6) died on day 18 (Figure 2A; Table 1). This animal had a transient low-level ZEBOV viraemia on day 6 and had cleared the ZEBOV infection by day 10 (Figure 2B). Furthermore, the animal never developed clinical symptoms consistent with severe ZEBOV HF, and organ infectivity titration showed no evidence of infectious ZEBOV in any of the tissues surveyed at post-mortem. Pathology results showed that this macaque died from disseminated septicaemia and peritonitis caused by Streptococcus pneumoniae as demonstrated by immunohistochemistry (unpublished data). The source of the bacterial infection is unknown. Both monkeys treated with the VSV control vectors (subjects c1 and c2) developed severe symptoms over the disease course with plasma viraemia titres in excess of 1 × 106 pfu/ml on day 6, macular rash (Figure S2) evident by day 7, and death on day 8 after ZEBOV challenge (Figure 2A; Table 1) with peak viraemia titre of >1 × 108 pfu/ml (Figure 2B). In addition, all animals were also tested for VSV viraemia using RT-PCR (unpublished data). In accordance with our previous results [9,10], VSV RNA was detected in most immunized animals only at day 3 post-immunization indicating transient viraemia of the vaccine vector. There was no correlation between VSV viraemia and survival. Table 1 Clinical Findings Figure 2 Survival and Plasma Viraemia for Rhesus Monkeys Given Post-Exposure Treatment for ZEBOV Infection (A) Kaplan-Meier survival curves for animals treated with ∼2 ×107 pfu of VSVΔG/ZEBOVGP (subjects 1 to 8, solid line) or VSV control vectors (subjects c1 and c2, dotted line) 20–30 min after i.m. challenge with 1,000 pfu of ZEBOV. (B) Plasma viraemia of animals treated with VSVΔG/ZEBOVGP or VSV control vectors 20–30 min after i.m. challenge with 1,000 pfu of ZEBOV. Viraemia was determined by plaque assay at indicated time points. The asterisk indicates that on day 8 post-challenge viraemia levels were only determined for the control animals (subjects c1 and c2). Plasma viraemia levels at day 6 post-ZEBOV challenge could be separated into three different groups. Control animals, which received VSV control vectors (black square), developed high plasma viraemias (>6 log10 pfu/ml). Animals treated with VSVΔG/ZEBOVGP, which developed fulminant EBOV HF and succumbed to ZEBOV challenge (orange square), developed moderate plasma viraemias (∼4–6 log10 pfu/ml), while animals treated with VSVΔG/ZEBOVGP, which survived (green square), had low plasma viraemias (≤1.4 log10 pfu/ml). Subject 6 did not develop fulminant disease consistent with EBOV HF and succumbed on day 18 from a secondary bacterial infection. All four animals that survived the ZEBOV challenge (subjects 1, 2, 5, and 7), and the animal that survived until day 18 (subject 6), developed ZEBOV-specific humoral immune responses with low titre IgM antibodies detected on days 6–14 (subjects 1, 5, and 7) (Figure 3A) and moderate IgG antibody titres detected on days 10–22 (subjects 1, 2, 5, 6, and 7) (Figure 3B). Neutralizing antibody titres to ZEBOV (1:80) were detected on days 14–37 after challenge in all four animals that survived the ZEBOV challenge (subjects 1, 2, 5, and 7) and the animal that survived until day 18 (subject 6) (Figure 3C). Humoral immune responses could not be detected in any of the non-survivors although these animals lived until day 9 and 10 post-challenge, which was sufficient to mount detectable IgM and IgG responses in the surviving animals. Figure 3 Serological Response Profile for Rhesus Monkeys Given Post-Exposure Treatment for ZEBOV Infection IgM (A), IgG (B), and development of EBOV-neutralizing antibodies (C) in sera of animals treated with 2 × 107 pfu of VSVΔG/ZEBOVGP 20–30 min after i.m. challenge with 1,000 pfu of ZEBOV. We also evaluated changes in populations of peripheral blood mononuclear cells during the course of the study to identify any differences between the rhesus monkeys treated with the VSVΔG/ZEBOVGP vector and the controls. A rapid loss of CD4+ lymphocytes, CD8+ lymphocytes, and NK cells has been reported during ZEBOV infection of nonhuman primates [28]. In this study, we also detected a decline in the circulating CD4+ and CD8+ (2%–10% decrease) lymphocyte populations on day 6 in most of the animals regardless of treatment or outcome with a 7%–22% decrease and 2%–10% decrease in cell numbers observed, respectively (Table 1). However, the percentage of NK cells did not drop in any of the animals treated with VSVΔG/ZEBOVGP vector on day 6, but markedly increased. Interestingly, a sharp decline in NK cell number (10% decrease) was observed on day 10 in one of the animals treated with the VSVΔG/ZEBOVGP vector. Similarly, a marked increase in B cells was noted for all animals regardless of treatment or outcome on day 6, followed by a decline in B cell number on day 10. Discussion Although no EBOV vaccine is currently licensed for human use, recent advances have been made and efficacy studies in nonhuman primates with several platforms have been encouraging [6,7,9]. Far less progress has been made in developing treatment interventions for EBOV infections [5,13,14]. Thus, there is clearly a need to develop effective strategies to respond to future EBOV outbreaks in Africa and to counter acts of bioterrorism using EBOV. Additionally, the potential EBOV exposure involving a researcher at a United States Army laboratory [16] and the unfortunate death of a Russian scientist after an accidental exposure to EBOV [15], underscore the need for medical countermeasures for post-exposure prophylaxis. Recently, a post-exposure strategy to mitigate the coagulation disorders that typify filoviral infections improved survival from 0% to 33% in the rhesus macaque model of ZEBOV HF [11]. Here, we show a significant advance in treating EBOV infections. Our data clearly demonstrate the efficacy of the VSV-based EBOV vaccine vector in post-exposure treatment in three relevant animal models. In the mouse model it was possible to protect all animals following challenge with treatment as late as 24 h post infection. It is known from previous data that treatments and vaccines given to mice are more effective than seen in guinea pigs and nonhuman primates [1,2,13]. However, in this case it was possible to protect over 50% of guinea pigs and 50% of nonhuman primates from uniformly lethal ZEBOV challenge. It should be noted that mice received about 10 or 100 times more vaccine per weight than guinea pigs and nonhuman primates, respectively. Thus, it is possible that further optimization of dosing strategies could improve the results. The rhesus macaques that survived infection all controlled the virus within the first 6 d of infection. The data clearly show that moderate or high-level viraemia on day 6 invariably resulted in a fatal outcome (Figure 2). In the current study, we can conclude that neutralizing antibodies were not essential for infection control (Figure 3) since they were not detected until after the animals had cleared the EBOV infection. Circulating CD4+ and CD8+ T cells were reduced in number in all animals regardless of treatment (Table 1); this indicates that the initial control of infection may not require classical T-cell responses. The time course for EBOV HF in rhesus macaques is very short (∼8 d) and therefore, CD8+ cytotoxic T-cell responses are very unlikely to be involved in the control of the infection because the cell numbers of specific responding cells could not have peaked until after the infection was controlled. The primary immune correlate of protection seems to be the rapid development of non-neutralizing antibody that was only seen in the protected animals (Figure 3). This, coupled with the NK-cell increase in the VSVΔG/ZEBOVGP-treated animals, may have resulted in significantly enhanced killing of virus-infected primary target cells and, consequently, elimination of the ZEBOV infection. An important role of NK cells for protection has also been described for immunization with virus-like particles [29]. Clearly, the adaptive response is essential to promote survival as animals immunized with the control VSV-based vaccines succumbed to the ZEBOV challenge (Figure 2, Table 1). Both control animals died on day 8, which is the historical mean for rhesus monkeys infected by the same route and dose with this seed stock (historical n = 23). However, other mechanisms probably contribute as well. Recently, Noble and colleagues described a new paradigm for an interfering vaccine in which one of the antiviral mechanisms of action is intracellular interference with the replication of the lethal wild-type virus [30]. In the current study, the VSV vectors exploit the EBOV GP, which largely determines host cell tropism and mediates viral entry [31]. We have demonstrated that the VSV vectors expressing the ZEBOV GP will infect the same cells as wild-type ZEBOV in vitro [8]. Also, the VSVΔG/ZEBOVGP vectors replicate significantly faster than wild-type ZEBOV [8]. Therefore, it is possible that these vectors compete with ZEBOV through viral interference. Clearly, even mild to moderate inhibition of ZEBOV replication may delay the course of infection and tip the balance in the favor of the host. VSV has been shown to be a potent inducer of the innate and adaptive immune system [32–34]. In contrast, EBOV has acquired mechanisms to counteract the innate immune responses of the host at different levels [1,2,35]. The virion protein (VP) 35 of ZEBOV functions as an inhibitor of type I interferon production by blocking the activation of IRF-3 [36–38]. In addition to VP35, the ZEBOV protein VP24 functions as an inhibitor of type I interferon signaling by blocking nuclear accumulation of activated STAT-1 [35,39]. Recently, it was suggested that VP24 blocks the downstream signaling cascades activated by type I interferon by inhibiting the phosphorylation of p38 [40]. Therefore, treatment with the VSV vectors might induce or boost the innate immune response in the host, and thus, counteract the immune inhibitory effect of EBOV. In this case, the host will mount a nonspecific innate immune response allowing for time to develop a specific adaptive response that can overcome the EBOV infection and again tip the balance in favor of survival of the host. In a historical context, it is important to note that the mechanism for post-exposure protection of humans against smallpox and rabies are also not fully understood. For post-exposure treatment of rabies, levels of neutralizing antibodies have been used as a measure of protection. However, several studies of HIV-infected patients with likely or proven exposure to rabies showed that these patients failed to develop neutralizing antibodies after post-exposure rabies vaccination, yet there were no reports of death of these patients attributed to rabies [41,42]. Moreover, studies in mice suggest that cell-mediated immunity may play an essential role in post-exposure protection [43]. In the case of smallpox, post-exposure protection is presumed to be due in part to differences in the route of exposure and growth kinetics of the wild-type variola virus versus the vaccinia vaccine [20]. Briefly, infection with variola usually starts in the upper and lower respiratory tract with subsequent spread to lymphoid tissues. Thus, the natural variola infection proceeds much slower than post-exposure i.m. vaccinia vaccination, which bypasses the respiratory tract infection. In addition, it appears that vaccinia has a shorter incubation period than variola virus resulting in a more rapid development of cell-mediated immunity and neutralizing antibody. However, a recent study using monkeypox in the macaque model demonstrated better results with antiviral therapy than post-exposure vaccination [44]. Post-exposure treatment with the VSV-based MARV vaccine vector against MARV challenge was more potent and resulted in complete survival, no disease, and undetectable viraemia [12]. The development of symptoms and viraemia in MARV-infected rhesus monkeys is delayed compared with ZEBOV [12,45], which may explain the difference in efficacy in post-exposure treatment with the VSV-based vectors. The efficacy of the VSV-based EBOV vector in post-exposure treatment might be increased by a higher treatment dose or multiple treatments over a longer period of time as is being done in post-exposure treatment of rabies [46]. Alternatively, combination therapy should be considered to increase therapeutic efficacy. In the case of EBOV, post-exposure treatment with the VSV-based EBOV vector could be combined with the previously published post-exposure strategy to mitigate the coagulation disorders [11]. Nevertheless, the VSV-based ZEBOV vaccine currently provides the most effective and promising single treatment strategy for EBOV HF. It is likely that the mechanism of protection by the VSV-based vaccine is multifactorial; while NK cells and antibody responses appear to be important to survival, viral interference and innate immune response are almost certainly essential in delaying the progression of the ZEBOV infection and extending the window for the adaptive response to become functional. Post-exposure treatment is particularly suited for use in accidentally exposed individuals and in the control of secondary transmission during naturally occurring outbreaks or deliberate releases. Our results also suggest that this VSV platform might be even more beneficial as a fast-acting single-shot preventive vaccine. Finally, this system also provides an excellent opportunity to study the fundamental mechanisms that lead to such devastating disease following infection with ZEBOV. Supporting Information Figure S1 Weight Loss of Mice and Guinea Pigs Given Post-Exposure Treatment for ZEBOV Infection (A) Mice (groups of five animals) were infected with 1,000 LD50 of MA-ZEBOV by i.p. injection. At various times points 24 h prior to challenge (○), 30 min after challenge (♦), or 24 h after challenge (▵) they were treated with 2 ×105 pfu of VSVΔG/ZEBOVGP by i.p. injection. The controls (▪) were left untreated and all died. All treated animals survived the challenge. (B) Guinea pigs (groups of six animals) were infected with 1,000 LD50 of GA-ZEBOV by i.p. injection. At various times points 24 h prior to challenge (○), 1 h after challenge (♦), or 24 h after challenge (▵) they were treated with 2 ×105 pfu of VSVΔG/ZEBOVGP by i.p. injection. The controls (▪) were left untreated and all died. †, animals that succumbed to infection in the treated groups. (980 KB TIF) Click here for additional data file. Figure S2 Clinical Symptoms Macular rash covering the inguinal region and inner leg of a VSVΔG/ZEBOVGP-treated macaque (subject 8) that succumbed 10 d after ZEBOV challenge. (5.1 MB TIF) Click here for additional data file. Accession Numbers The GenBank (http://www.ncbi.nlm.nih.gov/Genbank) accession number for the ZEBOV Mayinga strain is AF272001; the accession number for the MARV Musoke strain is Z12132.

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Antigenic Subversion: A Novel Mechanism of Host Immune Evasion by Ebola Virus

Gopi Mohan, Wenfang Li, Ling Ye … (2012)

Introduction Ebola virus (EBOV) is an enveloped single-stranded negative-sense RNA virus in the order Mononegavirales, which along with the Marburg virus (MARV) forms the Filovirus family. EBOV is the etiologic agent of Ebola Hemorrhagic Fever (EHF), a highly lethal hemorrhagic fever with up to 90% mortality [1]. Since its discovery in 1976, EBOV has caused sporadic outbreaks in Sub-Saharan Africa with death tolls in the hundreds. Interestingly, while filoviruses have been only recently discovered, they are one of the few non-retrovirus RNA paleoviruses identified in mammalian genomes, suggesting an ancient relationship with mammals [2], [3]. Growing evidence suggests that bats are the natural reservoir of EBOV in the wild today [4]–[6]. Current treatment for Ebola hemorrhagic fever is purely supportive, and the lack of effective interventions underscores the importance of developing a broadly-protective vaccine that confers long-lasting immunity. The ability to develop such a vaccine is critically dependent on our understanding of the mechanisms by which EBOV suppresses, distracts, or otherwise evades the host immune response [7]. One widely hypothesized immune evasion mechanism employed by Ebola virus is secretion of a truncated viral glycoprotein by EBOV infected cells. The EBOV surface glycoprotein (GP1,2) mediates host cell attachment and fusion, and is the primary structural component exposed on the virus surface. For this reason, GP1,2 is the focus of most EBOV vaccine research, and it is generally accepted that a robust anti-GP1,2 antibody response is crucial for protection against lethal EBOV challenge [8]. EBOV GP1,2 forms trimeric spikes on virion surfaces similarly to influenza HA and HIV Env [9]. Also like HA and Env, GP is first synthesized as an uncleaved precursor (GP0) which is then cleaved in the Golgi complex by the protease furin [10] into two functional subunits: The N-terminal GP1 subunit contains the putative receptor-binding domain (RBD), and the C-terminal GP2 subunit contains the fusion apparatus and transmembrane domain. GP1,2 is encoded in two disjointed reading frames in the virus genome. The two reading frames are joined together by slippage of the viral polymerase at an editing site (a tract of 7-A's) to insert an 8th A, generating an mRNA transcript that allows read-through translation of GP1,2 [11], [12]. However, only about 20% of transcripts are edited, while the remaining 80% of unedited transcripts have a premature stop codon, resulting in synthesis of a truncated glycoprotein product (sGP) which is secreted in large quantities into the extracellular space. Though its production is conserved in all EBOV species, there has been considerable debate regarding the function of sGP. Unlike GP1,2, sGP forms homodimers and appears to have some intrinsic anti-inflammatory activity [13]–[17]. The recent finding that EBOV quickly mutates to synthesize primarily GP1,2 in cell culture, while this mutant virus reverts to a primarily sGP-producing phenotype in vivo, suggests an important role for sGP in virus survival within the host [18]. Because sGP shares over 90% of its sequence with the N-terminal region of GP1,2, it was initially hypothesized that sGP functions as a decoy for anti-GP1,2 antibodies. Early efforts to identify such activity yielded mixed results, and the observation that antibodies often do not cross-react between sGP and GP1,2 had cast doubt on this hypothesis [19]–[23]. Furthermore, recent studies demonstrated that immunization against GP1,2 elicits antibodies largely against epitopes not shared with sGP [24]–[27]. However, most of these studies investigated monoclonal antibodies from animals immunized with vaccines containing or expressing primarily GP1,2, which does not represent the state of natural infection. Of note, one early study examined monoclonal antibodies from mice immunized with a Venezuelan equine encephalitis replicon that produces both GP1,2 and sGP, and found that many of these antibodies cross-reacted between GP1,2 and sGP [28]. Further, monoclonal antibodies isolated from human EHF survivors have been shown to preferentially react with sGP [19]. These studies suggest that sGP may play an important role in altering the host antibody response. In this study, we demonstrate that sGP induces a host antibody response that focuses on epitopes it shares with GP1,2, thereby allowing it to bind and compete for anti-GP1,2 antibodies. We describe a mechanism that we term “antigenic subversion”, which is distinct from previously proposed “decoy” mechanisms in which secreted glycoprotein simply passively absorbs anti-glycoprotein antibodies. Importantly, we demonstrate that sGP can also subvert an existing anti-GP1,2 immune response that was only weakly cross-reactive with sGP. Antigenic subversion represents a novel host immune evasion mechanism that has important implications for EBOV vaccine design, and may shed light on how the virus survives in its natural reservoir. Results Immunogenicity of EBOV GP Editing Site Mutant DNA Vaccines We first generated EBOV GP constructs to individually express GP1,2 and sGP. In natural infection, EBOV directs the synthesis of sGP and GP1,2 through differentially edited mRNA transcripts (Fig. 1A). However, it has been observed that DNA-dependent RNA polymerases (DDRP) do not edit with the same efficiency as the EBOV RNA polymerase [12]. Furthermore, in addition to polymerase slippage, it is possible that the 7-A editing site can also serve as a premature poly-adenylation signal, as well as a ribosomal slippage signal [29]–[31]. We thus generated a panel of EBOV GP editing site mutants in order to control the levels of sGP and GP1,2 expression (Fig. 1B). GP-8A was made by inserting an 8th A into the wild type (GP-7A) editing site, resulting in GP1,2 as the dominant gene product. Silent A→G mutations were introduced into the GP-8A editing site to ablate transcriptional slippage, resulting in GP1,2Edit, that expresses GP1,2 as the sole gene product. The same mutations were also introduced into GP-7A to generate sGPEdit, that expresses sGP as the sole gene product. These constructs were subcloned into a mammalian expression vector (pCAGGS) and protein expression was examined in both HeLa cells (Fig. 1C) and 293T cells (data not shown). Cells transfected with GP-8A and GP1,2Edit expressed GP1,2 intracellularly and on their surfaces, and secreted GP1,2 into the supernatant through previously characterized TACE-dependent cleavage [32]. Interestingly, GP1,2Edit produced higher amounts of GP1,2 than GP-8A. GP-7A and sGPEdit expressed high levels of sGP, which was secreted efficiently into the supernatant. GP1,2 expression by GP-7A was undetectable, likely because of minimal DDRP-mediated editing [12]. These expression experiments demonstrate that mutation of the editing site has a significant effect on GP expression. 10.1371/journal.ppat.1003065.g001 Figure 1 Diagram of EBOV RNA editing and construction of EBOV GP mutants. (A) Schematic diagram of GP1,2 and sGP. Membrane-bound GP1,2 is encoded in the EBOV genome in two disjointed reading frames. The GP editing site is a tract of 7 A's approximately 900 nucleotides downstream of the start codon. Slippage of EBOV RNA-dependent RNA polymerase at the editing site results in insertion of an 8th-A which brings the two GP reading frames in register resulting in read-through translation of full-length membrane-bound trimeric GP1,2. Unedited transcripts contain a premature stop codon and produce truncated dimerized sGP. (B) EBOV GP and editing site mutants. Mutated nucleotides are shown in red and the primary gene products expressed by these constructs are also listed. (C) Expression of EBOV GP by wild type and mutant DNA constructs. HeLa cells were transfected with the wild type GP or editing site mutant constructs and GP expression was assayed by Western blot at 48 h post-transfection. We next investigated the immunogenicity of editing site mutant DNA vaccines. Female BALB/c mice were immunized with GP1,2 or sGP-producing constructs (Fig. 2A). Mice immunized with sGPEdit, GP-7A, and GP-8A constructs developed similar titers of anti-GP1,2 antibodies as measured by ELISA, while mice immunized with GP1,2Edit developed four-fold higher titers of anti-GP1,2 antibodies (Fig. 2B). Mice immunized with constructs expressing predominantly sGP (GP-7A and sGPEdit) developed much higher titers of anti-sGP antibodies than mice immunized with constructs expressing predominantly GP1,2 (GP-8A or GP1,2Edit) (Fig. 2C). As shown in Fig. 2D, GP1,2-immunized mice developed much higher titers of GP1,2-binding antibodies than sGP-binding antibodies. On the other hand, sGP-immunized mice developed much higher titers of sGP-binding antibodies than GP1,2-binding antibodies, despite the fact that sGP shares roughly 95% of its linear sequence with GP1,2. These results suggest that in sGP-immunized animals, either many sGP-binding antibodies are directed against conformational epitopes not shared with GP1,2, or they are directed against shared epitopes that are inaccessible in GP1,2. 10.1371/journal.ppat.1003065.g002 Figure 2 Immunogenicity of EBOV GP editing site mutants. (A) Immunization study design. Female BALB/C mice were immunized with the four editing site mutant constructs in the pCAGGS vector. Mice were vaccinated IM with 50 µg of DNA (25 µg/leg) according to the schedule shown. (B) Antibody response against GP1,2. (C) Antibody response against sGP. The levels of antibody response induced by EBOV GP DNA constructs in mice were measured by ELISA using His-GP1,2 or His-sGP as coating antigen. Antibody concentration was determined from a standard curve and expressed as µg/mL of anti-GP IgG. Asterisks indicate statistically significant difference between groups and P-values are given in red. (D) Comparison of antibody levels against GP1,2 and sGP induced by each EBOV GP DNA construct. Average titers of anti-GP1,2 (blue) and anti-sGP (red) antibodies within immunization groups are shown for comparison of the GP isoform reactivity profiles both within and between immunization groups. Asterisks indicate statistically significant differences between anti-GP1,2 and anti-sGP titers within groups, as measured by paired, two-tailed Student's t-test (* = p<0.05, ** = p<0.001). sGP Can Compete for Binding of Anti-GP1,2 Antibodies Induced by sGP but not by GP1,2 Given that animals immunized by GP1,2 or sGP develop antibodies that preferentially bind to different GP isoforms, we performed Western blot analysis to determine if there is a difference in the linear epitopes targeted by antibodies in GP1,2 versus sGP-immunized mice. As shown in Fig. 3A, antisera from GP1,2-immunized mice reacted strongly with GP1,2 but only weakly with sGP. On the other hand, antisera from sGP-immunized mice reacted strongly with sGP, but only weakly with GP1,2. This suggests that most linear epitopes targeted by anti-GP1,2 antibodies from GP1,2-immunized mice are unshared with sGP. To investigate whether the GP1,2-binding and sGP-binding antibodies in immunized mice were cross-reactive between the two GP isoforms or were separate populations of antibodies, we performed a competition ELISA to determine if sGP could compete with GP1,2 for GP1,2-binding antibodies (Fig. 3B). Similar to the Western blot data, sGP was unable to compete for binding of anti-GP1,2 antibodies from GP1,2 immunized mice (Fig. 3C). On the other hand, sGP was able to efficiently compete for anti-GP1,2 antibodies from sGP-immunized mice. As expected, GP1,2 was able to compete with itself in all groups (Fig. 3D). Furthermore, we observed an identical reactivity pattern with native membrane-anchored EBOV GP1,2 using a cell surface competition ELISA (Supplemental Fig. S1). We further examined the ability of the two GP isoforms to compete with each other for antibodies by performing competition immunoprecipitation. Purified GP1,2 in the presence of sGP at varying molar ratios was immunoprecipitated with antiserum from GP1,2-immunized or sGP-immunized mice, and analyzed by Western blot using a polyclonal rabbit antibody that reacts with both GP isoforms. Antiserum from GP1,2-immunized mice precipitated both GP1,2 and sGP, and increasing concentrations of sGP did not attenuate the amount of GP1,2 signal (Fig. 3E), suggesting the presence of two separate populations of antibodies that do not cross-react between GP1,2 and sGP. However, while antiserum from sGP-immunized mice also precipitated both GP1,2 and sGP, increasing concentrations of sGP significantly attenuated the amount of GP1,2 precipitated (Fig. 3F), indicating that GP1,2-reactive antibodies in these mice are cross-reactive with sGP. As a control, addition of recombinant HA had no effect on the amount of GP1,2 precipitated by either antiserum group. Taken together, these data suggest that anti-GP1,2 antibodies induced by GP1,2 are directed primarily against epitopes not shared between GP1,2 and sGP, whereas such antibodies induced by sGP are directed against epitopes shared between GP1,2 and sGP. 10.1371/journal.ppat.1003065.g003 Figure 3 Antiserum from mice immunized against GP1,2 or sGP display different reactivity patterns. (A) Detection by Western blot of antibodies against GP1,2 and sGP from immunized mice. 50 ng of purified His-sGP and His-GP1,2 were run by SDS-PAGE under denaturing conditions and probed with 1∶1000 pooled GP1,2Edit or sGPEdit antisera followed by blotting with HRP-conjugated goat anti-mouse IgG. (B) Schematic of competition ELISA. Wells were coated with GP1,2 and incubated with pooled antisera as well as increasing concentrations of competing antigen (sGP or GP1,2) to compete for antibodies. After two hours, plates were washed and then incubated with HRP-conjugated secondary antibody followed by addition of substrate to develop color. (C, D) Competition ELISA. Antisera from mice immunized with sGPEdit, GP-7A, GP-8A, and GP1,2Edit were diluted to give similar anti-GP1,2 signal. Diluted antiserum was mixed with increasing quantities of purified His-sGP (C) or His-GP1,2 (D) and incubated in His-GP1,2 coated wells and developed as described above. Experiments were performed in duplicate and repeated at least three times, with representative results shown. (E, F) Competition Immunoprecipitation. Pooled antisera from GP1,2Edit-immunized mice (E) or sGP-immunized mice (F) were incubated with no GP, purified sGP or GP1,2 alone, or with fixed GP1,2 and increasing concentrations of sGP to compete for anti-GP1,2 antibodies. GP1,2 was incubated with recombinant HA as a negative control. The upper panel for the sGPEdit antisera shows the GP1,2 portion of the blot at a longer exposure time to show the attenuation of signal with increasing sGP concentration. Results are representative of three independent experiments. sGP Differentially Interferes with Antibody-mediated Viral Neutralization by Antisera from sGP and GP1,2 Immunized Mice We further investigated whether there was a difference in the ability of antisera from the immunization groups to neutralize EBOV GP1,2-mediated virus infection, and whether sGP could interfere with antibody-mediated neutralization. Pseudoviruses were generated using an Env-deficient HIV backbone pseudotyped with Zaire EBOV GP1,2. In order to achieve consistent neutralization, we pooled sera from the four highest responders among GP1,2-immunized animals and among sGP-immunized animals. Antisera from both groups were able to effectively neutralize pseudoviruses as measured by a luciferase reporter assay (Fig. 4A), although antisera from GP1,2-immunized mice exhibited more potent neutralizing activity than antisera from sGP-immunized mice, probably due to higher overall anti-GP1,2 titer. To determine if sGP interferes with neutralization, we used an antiserum dilution corresponding to 80% neutralizing activity in each group and preincubated antisera with different amounts of sGP. Consistent with the competition ELISA results, sGP was able to completely attenuate neutralizing activity of antisera from sGP-immunized mice, while it had no effect on neutralizing activity of antisera from GP1,2-immunized mice (Fig. 4B). Purified influenza HA was used as a control and had no effect on neutralizing activity of either antiserum group. Similar results were observed when we used an antiserum dilution corresponding to 50% neutralizing activity (Supplemental Fig. S2). These data confirm that sGP can compete with GP1,2 for anti-GP1,2 antibodies and interfere with antibody-mediated neutralization, but can only do so in animals that have been exposed to sGP. 10.1371/journal.ppat.1003065.g004 Figure 4 Interference with antibody-dependent neutralization by sGP. (A) Neutralization of EBOV GP pseudovirus. Neutralizing activity of antisera was determined by incubating 500 pfu of GP1,2-pseudotyped virus with dilutions of pooled GP1,2-immunized (Blue), sGP-immunized (Red), and empty pCAGGS vector-immunized (black) antisera. Neutralization was measured as decrease in luciferase expression compared to virus-only controls after 48 h. (B) Interference of EBOV GP pseudovirus neutralization by sGP. The ability of sGP to interfere with antibody-dependent neutralization was determined by allowing sGP to compete with GP1,2 pseudotyped viruses for anti-GP1,2 antibodies. Pooled GP1,2-immunized (blue) and sGP-immunized (red) antisera were fixed at the dilution corresponding to 80% neutralization. Antisera was co-incubated with increasing dilutions of His-tagged sGP (solid markers) or His-tagged influenza PR8 HA (open markers), and rescue of infectivity was measured as described in methods. Anti-GP1,2 and Anti-sGP Antibodies Induced by Different GP Isoforms Exhibit Similar Average Affinity The inability of sGP to compete with GP1,2 for antibodies from GP1,2-immunized mice was intriguing considering that GP1,2 shares almost half of its ectodomain sequence with sGP. We reasoned that some of these antibodies may be directed solely against GP1,2 epitopes not shared with sGP, while other antibodies may be directed against shared epitopes, but preferentially bind GP1,2 because of conformational differences between the two GP isoforms resulting from tertiary and quarternary structure and steric shielding. To address this possibility, we used quantitiative ELISA to determine the relative titers and estimate the average affinity of antibodies from GP1,2 and sGP-immunized animals for GP1,2 and sGP. We individually examined purified polyclonal IgG from the five highest responders in GP1,2-immunized and sGP-immunized groups, and calculated the apparent dissociation constant (Kd) of anti-GP1,2 and anti-sGP antibodies. This apparent Kd was calculated by Scatchard analysis as described elsewhere [33], [34] and represents an estimate of the average affinity of anti-GP antibodies, with lower apparent Kd correponding to higher average affinity. Consistent with above ELISA data (Fig. 2D), mice immunized against GP1,2 had higher titers of anti-GP1,2 antibodies than anti-sGP antibodies (Fig. 5A). However, there was no measurable difference in the apparent Kd's of GP1,2-binding vs. sGP-binding antibodies (Fig. 5B), indicating that preferential binding of antibodies from these animals to GP1,2 is not due to affinity differences for different GP isoforms. In mice immunized against sGP we again observed very high titers of anti-sGP antibodies, and very low levels of anti-GP1,2 antibodies. However, those antibodies that did bind to GP1,2 appeared to have modestly lower Kd (higher average affinity) than did sGP-binding antibodies (Fig. 5B). Future studies with monoclonal antibodies directed against epitopes shared between sGP and GP1,2 will provide further information on whether specific antibodies bind to the two GP isoforms with different affinities. Nonetheless, the present data provide evidence that differences in affinity are not responsible for antibodies from GP1,2 and sGP-immunized mice reacting preferentially with different GP isoforms. 10.1371/journal.ppat.1003065.g005 Figure 5 Comparison of binding affinity of GP1,2-immunized versus sGP-immunized antisera for sGP and GP1,2. (A) Determining apparent Kd value of antibodies from immunized mice for GP1,2 and sGP. Antiserum from five mice immunized against GP1,2 and five mice immunized against sGP were individually analyzed by quantitative ELISA using GP1,2 (blue) or sGP (red) as coating antigen. Scatchard analysis was used to calculate apparent dissociation constants (Kd). (B) Comparison of antibody affinity for GP1,2 and sGP. Comparison of apparent Kd's of GP1,2-immunized and sGP-immunized polyclonal antisera for sGP (red) and GP1,2(blue) was determined by nonlinear regression analysis of Scatchard plots. Kd's for sGP and GP1,2 were calculated for five individual mice in each group and values for the same animal are connected by a black line. Expression of GP1,2 in the Context of sGP Allows sGP to Compete for Anti-GP1,2 Antibodies The secretion of surface glycoproteins as a mechanism of absorbing antiviral antibodies has been hypothesized before for several viruses including vesicular stomatitis virus (soluble G) and respiratory syncytial virus (secreted G) [35], [36]. It has been demonstrated that RSV secreted G can absorb anti-G antibodies and interfere with both neutralization and antibody-dependent cell-mediated virus clearance. However, we observed that EBOV sGP can only compete for anti-GP1,2 antibodies in mice immunized against sGP. This led us to hypothesize that sGP may serve a role in altering the repertoire of epitopes against which the host immune response is directed, in order to divert the host immune response towards epitopes shared between sGP and GP1,2. To test this hypothesis, we vaccinated mice with a 3∶1 ratio of sGPEdit∶GP1,2Edit (Fig. 6A) to simulate antigen expression during EBOV infection. Control groups were immunized with either sGPEdit or GP1,2Edit plus empty pCAGGS vector to keep the total amount of DNA constant. As a proxy for in vivo antigen expression, HeLa cells were transfected with corresponding ratios of sGPEdit, GP1,2Edit, and pCAGGS. As measured by Western blot analysis, the levels of sGP and GP1,2 expression in both lysate and culture supernatant of cells co-transfected with sGPEdit and GP1,2Edit were similar to cells transfected with sGPEdit or GP1,2Edit alone (Fig. S3). All immunization groups generated similar titers of anti-GP1,2 antibodies (Fig. 6B). However, when we performed a competition ELISA using antisera from sGPEdit+ GP1,2Edit-immunized mice, sGP was able to compete with GP1,2 for over 50% of the anti-GP1,2 antibodies (Fig. 6C). Mice immunized with GP1,2Edit+vector or sGPEdit+vector displayed the same serum reactivity patterns we had observed previously in mice immunized against only one of the GP isoforms. Further, after boosting mice a second time, almost 70% of GP1,2-antibodies in week 12 antisera from sGPEdit+ GP1,2Edit-immunized mice were absorbed by sGP. Interestingly, in mice immunized with lower ratios of sGPEdit∶GP1,2Edit, significant sGP cross-reactivity was also observed, with almost 70% of anti-GP1,2 antibodies being susceptible to competition in mice immunized with a 1∶1 ratio of sGP∶GP1,2, and about 25% being susceptible to competition in mice immunized with a 1∶3 ratio of sGP∶GP1,2 (Figure S4). Similar results were also obtained with a competition immunoprecipitation assay. As shown in Fig. 6D, antiserum from sGPEdit+GP1,2Edit-immunized mice was able to precipitate both GP1,2 and sGP, but increasing concentrations of sGP attenuated the amount of GP1,2 precipitated. Furthermore, while sGPEdit+GP1,2Edit antiserum was able to effectively neutralize pseudovirus infectivity (Fig. 6E), the addition of exogenous sGP almost completely inhibited pseudovirus neutralization (Fig. 6F), indicating that sGP can effectively interfere with antibody mediated neutralization in these mice. Similar observations were also made at an antiserum concentration corresponding to 50% neutralization (Fig. S5). Taken together, these data confirm that sGP can direct the host antibody response to focus on epitopes shared between GP1,2 and sGP, thereby allowing sGP to compete for antibodies and interfere with antibody-mediated virus neutralization. Furthermore, the observation that sGP can compete for a greater proportion of GP1,2 antibodies from week 12 antisera compared to week 6 suggests that iterative exposure to sGP gradually drives the host to a dominantly sGP-reactive response. 10.1371/journal.ppat.1003065.g006 Figure 6 The effect of sGP on immune response when antigen exposure mimics natural infection. (A) Immunization study design. Female BALB/C mice were immunized IM with 50 µg of total DNA per immunization according to the schedule shown. Mice were immunized with a 3∶1 ratio of sGP Edit∶GP1,2 Edit in pCAGGS. Control groups were immunized with sGP Edit or GP1,2 Edit alone plus empty pCAGGS vector to keep total amount of immunizing DNA constant. (B) Comparison of antibody response against GP1,2. Mouse sera collected at week 6 were analyzed for anti-GP1,2 antibodies by ELISA using GP1,2 as coating antigen. (C) sGP competition ELISA. The ability of sGP to compete for anti-GP antibodies was determined by competition ELISA as in Figure 3B. Pooled antisera were analyzed from mice immunized with a GP1,2 Edit (blue), sGP Edit (red), or a 3∶1 ratio of sGP Edit∶GP1,2Edit (purple), and were diluted to give roughly equivalent anti-GP1,2 signal. Competition ELISA was performed from antisera collected at both week 6 (light color) and week 12 (dark color) according to the immunization schedule. (D) Competition immunoprecipitation. Pooled antisera from sGPEdit+GP1,2Edit-immunized mice were incubated with no GP, purified sGP or GP1,2 alone, or with fixed GP1,2 and increasing concentrations of sGP to compete for anti-GP1,2 antibodies. GP1,2 was incubated with recombinant HA as a negative control, and precipitated and analyzed as in Figure 3E,F. (E) Neutralization of EBOV GP pseudovirus. Neutralizing activity of antisera was determined by incubating 500 pfu of GP1,2-pseudotyped virus with dilutions of pooled sGP+GP1,2-immunized (red), or empty pCAGGS vector-immunized (black) antisera. Neutralization was measured as decrease in luciferase expression compared to virus-only controls. (F) Interference of EBOV GP pseudovirus neutralization by sGP. The ability of sGP to interfere with antibody-dependent neutralization was determined as in Figure 4B. Pooled sGP+GP1,2-immunized antisera were fixed at the dilution corresponding to 80% neutralization. Antisera were co-incubated with increasing dilutions of purified sGP (red) or purified influenza PR8 HA (blue), and rescue of infectivity was measured as described in methods. sGP Can Subvert the GP1,2-specific Antibody Response In order to test the hypothesis that expression of sGP can modulate the GP1,2-specific antibody response, we primed and boosted mice with either sGPEdit or GP1,2Edit, and then boosted again at week 10 with the opposite GP isoform (Fig. 7A). Control groups were boosted with the same GP isoform. As shown in Fig. 7B, anti-GP1,2 antibodies were induced in all groups at week 12. However, in mice immunized with GP1,2Edit and then boosted with sGPEdit, sGP was able to efficiently compete for anti-GP1,2 antibodies in competition ELISA (Fig. 7C). Furthermore, sGP was also able to efficiently compete for anti-GP1,2 antibodies from mice primed against sGPEdit and boosted with GP1,2Edit. We next investigated whether sGP is able interfere with virus neutralization by sera from cross primed and boosted mice. As shown in Fig. 7D, sGP was able to interfere with neutralization only from animals primed against sGP and boosted with GP1,2. On the other hand, antisera from animals primed against GP1,2 and boosted with sGP maintained their neutralizing activity in the presence of sGP. To further probe this observation, we compared the antisera titers corresponding to 50% neutralizing activity (NT50) in groups before (week 6) and after (week 12) boosting with the opposite GP isoform. As shown in Fig. 7E, neutralizing activity is not boosted by immunization with the opposite GP isoform. Thus, it appears not only that sGP can overwhelm the GP1,2-specific response, but also that it only boosts non-neutralizing antibodies induced by GP1,2. The observation that sGP can alter the reactivity profile of the anti-GP1,2 response has important implications for EBOV vaccinology, since during a infection, sGP could subvert the immune response of a previously vaccinated individual if the virus is not cleared rapidly. 10.1371/journal.ppat.1003065.g007 Figure 7 Ability of sGP to divert antibody responses against GP1,2. (A) Immunization study design. Female BALB/C mice were immunized IM with 50 µg of total DNA per immunization according to the schedule. Two groups of mice (n = 12) were primed and boosted as in previous experiments with either sGP Edit or GP1,2 Edit in pCAGGS vector. Each group was divided in two and subgroups were boosted at week 10 with either the same construct against which they had initially been immunized, or with the opposite editing site mutant construct. (B) Comparison of antibody response against GP1,2. Sera collected at week 12 were analyzed for antibodies against GP1,2 by ELISA using GP1,2 as coating antigen. (C) sGP competition ELISA. The ability of sGP to compete for anti-GP1,2 antibodies was determined by competition ELISA as described in Figure 3B. Pooled antisera were analyzed from mice immunized with sGP Edit and then boosted at week 10 with either GP1,2 Edit (red), or sGP Edit (purple), and from mice immunized with GP1,2 Edit and then boosted at week 10 with either GP1,2Edit (blue) or sGP Edit (green). All ELISA experiments were performed in duplicate at least three times and representative results shown. (D) Interference of EBOV GP pseudovirus neutralization by sGP. The ability of sGP to interfere with antibody-dependent neutralization was determined as in Figure 4B. Pooled sGP-primed, GP1,2-boosted (red) and GP1,2-primed, sGP-boosted (green) antisera were fixed at the dilution corresponding to 50% neutralization. Antisera were co-incubated with increasing dilutions of His-tagged sGP (solid markers) or His-tagged influenza PR8 HA (open markers), and rescue of infectivity was measured as described in methods. (E) Comparison of 50% neutralization titers. Antiserum titers corresponding to 50% pseudovirus neutralization activity (NT50) were calculated for week 6 (fine checkered) and week 12 (coarse checkered) mice. Error bars correspond to 95% confidence interval as determined by Student's t-test. Discussion The role of sGP in EBOV host immune evasion has not been clearly defined. In this study, we analyzed antibody responses in mice immunized against sGP, GP1,2, or both GP isoforms and present evidence that sGP serves to redirect the immune response towards epitopes that are either not present or inaccessible in GP1,2, or epitopes that are shared between the two GP isoforms, thereby allowing sGP to effectively absorb anti-GP1,2 antibodies. We term this phenomenon “antigenic subversion”, because it is distinct from previously proposed mechanisms in which sGP passively absorbs anti-glycoprotein antibodies. In antigenic subversion, the ability of sGP to absorb anti-GP1,2 antibodies is critically dependent on exposure to sGP during induction of the anti-GP1,2 immune response. In mice immunized against GP1,2 in the presence of sGP, an immunization strategy designed to simulate antigen exposure during natural infection, we observed that most resulting anti-GP1,2 antibodies were cross reactive with and thus susceptible to competition by sGP, even though the titers of anti-GP1,2 antibodies in these mice were similar to the titers in mice immunized against GP1,2 alone. On the other hand, in mice immunized against GP1,2 alone, we observed only low cross-reactivity of anti-GP1,2 antibodies with sGP, a finding consistent with previous studies, indicating that antibodies in these mice are largely directed against epitopes not shared with sGP [23], [24]. The model we propose for the mechanism of antigenic subversion by sGP assumes that before immunization, the host begins with a repertoire of naïve B-cells that recognize epitopes distributed throughout GP1,2 and sGP (Fig. 8A). However, because sGP is generated in much higher quantities than GP1,2, B-cells that recognize sGP epitopes and epitopes shared between sGP and GP1,2 are more likely to encounter their cognate antigens as compared with B-cells that recognize GP1,2-specific epitopes. Furthermore, as the sGP-reactive B-cell population expands, it will outcompete other B-cells for antigen and survival signals. Thus, the humoral response is skewed towards sGP, and epitopes of GP1,2 that are shared with sGP. Antigenic subversion represents a novel viral escape strategy that has some similarities to original antigenic sin (OAS). In classical OAS, initial exposure to a pathogen results in a population of memory B-cells that recognize antigens specific to that pathogen strain. Upon subsequent exposure to a different strain of the same pathogen, cross-reactive memory B-cells will respond preferentially, producing antibodies with high affinity to the initial pathogen which may not bind to the new strain as effectively [37], [38]. Furthermore, these memory B-cells can compete for antigen and survival signals with naïve B-cells that might otherwise produce higher affinity or more protective antibodies to the new strain. Similarly, overexpression by Ebola virus of sGP ensures that sGP-reactive B-cells preferentially expand and outcompete GP1,2-specific B-cells for antigen and survival signals, resulting in a suboptimal host response that is directed away from membrane-bound GP1,2 on the virion surface. However, unlike classical OAS, this process does not require temporal separation of antigen encounters, but can also occur during simultaneous exposure to two partly identical antigens. 10.1371/journal.ppat.1003065.g008 Figure 8 Proposed mechanism for antigenic subversion. Regions of GP1,2 that are shared with sGP are in red, while unshared epitopes are in green. B-cells are colored according to the regions of GP1,2 and sGP against which they react. (A) A naïve animal begins with B-cells that can potentially recognize epitopes distributed throughout GP1,2 and sGP. When sGP is expressed at much higher levels than GP1,2, as occurs during infection, those B-cells that recognize sGP epitopes, many of which are shared with GP1,2 (red regions of sGP and GP1,2) are preferentially activated and expanded compared to B-cells that recognize unshared epitopes of GP1,2 (green regions of GP1,2). Thus, sGP-reactive antibodies dominate the immune response. (B) Prior immunization by sGP. Because sGP shares over 90% of its linear sequence with GP1,2, animals primed with sGP generate anti-sGP antibodies, many of which are directed against epitopes shared with GP1,2. When these animals (or individuals who have previously been infected and recovered from EBOV infection) are boosted with GP1,2, sGP cross-reactive memory cells outnumber and express higher affinity receptors than naïve GP1,2 specific B-cells, resulting in preferential expansion of these sGP-cross-reactive B-cells and a predominantly sGP-reactive immune response. (C) Prior immunization by GP1,2. Priming naïve animals with GP1,2 results in antibodies largely against GP1,2 epitopes not shared with sGP, presumably due to the immunodominance and high accessibility of the GP1,2 mucin domain and shielding of shared epitopes. When these animals are boosted with sGP, or if they are infected with EBOV and do not have sufficiently high titers of anti-GP1,2 antibodies to clear the infection rapidly, memory B-cells that recognize shared epitopes encounter their cognate antigen and expand, while non-cross-reactive GP1,2-specific B-cells are not boosted, resulting in subversion of the host immune response towards sGP cross-reactivity. (D) Successful clearance of EBOV infection. In order to avoid sGP-mediated antigenic subversion, high enough titers of non-crossreactive anti-GP1,2 antibodies must be maintained to rapidly clear EBOV infection before subversion can occur. Our model for antigenic subversion can also explain how anti-GP1,2 antibodies from animals primed against sGP and then boosted with GP1,2 maintain cross-reactivity with sGP. In these animals, priming with sGP elicits antibodies against sGP epitopes, some of which are shared with GP1,2 (Fig. 8B). When these animals are boosted with GP1,2, memory B-cells that recognize shared epitopes vastly outnumber (and express higher affinity receptors than) the naïve B-cells that recognize unshared epitopes. Thus, the anti-sGP memory B-cells will be preferentially activated and expanded, boosting the anti-sGP response. This situation is analogous to one in which previously-infected individuals are vaccinated against GP1,2, and raises the possibility that immunizing such individuals may simply boost an already unprotective antibody response. While filovirus infection is rare, our findings suggest that it may be necessary to devise alternate strategies for immunizing previously-infected individuals in a way that specifically boosts the anti-GP1,2 response and avoids subversion. Perhaps the most striking finding in this study is that boosting GP1,2-immunized mice with sGP could effectively subvert the anti-GP1,2 response and render it susceptible to competition by sGP. We hypothesize that while the majority of B-cells activated in mice immunized against GP1,2 are directed against epitopes not shared with sGP (Fig. 8C), there is a small population of activated B-cells that react with sGP. This is supported by our observation that even though sGP cannot measurably compete in ELISA and immunoprecipitation for anti-GP1,2 antibodies from GP1,2-immunized mice, these mice still develop low titers of sGP-binding antibodies. When GP1,2-immunized mice are boosted with sGP, these sGP-reactive B-cells expand while the remaining GP1,2-specific B-cells that recognize unshared epitopes do not, shifting the anti-GP1,2 antibody response from mostly GP1,2-specific to mostly sGP-cross reactive. Furthermore, it is notable that neutralizing activity actually decreased after boosting with sGP, despite an increase in overall anti-GP1,2 antibodies. Thus, boosting with sGP only augmented non-neutalizing anti-GP1,2 antibodies that are highly susceptible to sGP competition, while the existing neutralizing antibodies previously induced by GP1,2 in these mice maintained resistence to sGP interference. This situation is analogous to one in which an individual is immunized against GP1,2 is subsequently infected with EBOV. If the individual is unable to rapidly clear the virus, the virus may replicate sufficiently to subvert the host immune response. Thus, it will be critical for vaccines to induce high enough titers of anti-GP1,2 antibodies to ensure that the virus is cleared before it is able to effect subversion (Fig. 8D). The inability of sGP to compete for anti-GP1,2 antibodies from GP1,2-immunized mice is consistent with a growing body of evidence pointing to the immunodominance of the GP1,2 mucin domain, a highly glycosylated region of GP1 not shared with sGP [24], [25]. This domain is thought to form a sterically bulky “cloak” that shields the putative receptor binding domain from host antibodies, as suggested for the HIV Env “glycan shield” [39]. The role that the mucin domain plays in host-pathogen interaction is complex and previous studies indicate that this region contains both neutralizing and infection-enhancing epitopes, and can mask epitopes on GP1,2 itself by steric occlusion [40], [41]. Furthermore, the mucin domain is the most divergent region of GP1,2 among EBOV strains, and is dispensible for GP1,2 mediated virus attachment and membrane fusion [42]–[44], strongly suggesting a role in protecting more functionally conserved regions of GP1,2 from immune attack. Because the linear sequence of sGP corresponds to the putative mucin-shielded receptor binding domain (RBD) of GP1, it is possible that sGP works together with the mucin domain so that host antibodies are directed either to shared epitopes that are sterically shielded in the GP1,2 trimer, or to the mucin domain itself, which is cleaved off in the host cell acidified endosome along with any bound antibodies [45], [46]. The possibility that GP1,2 epitopes shared with sGP may be shielded in the GP1,2 trimer is supported by our observation that very few anti-sGP antibodies in sGP-immunized mice cross-react with GP1,2 despite the fact that sGP shares over 90% of its linear sequence with GP1,2. Furthermore, antigenic subversion allows sGP to efficiently absorb those antibodies that do recognize unshielded and shared epitopes in GP1,2. The importance of sGP-mediated antigenic subversion to EHF pathogenesis remains to be elucidated. Passive immunization studies with polyclonal sera or monoclonal antibodies will reveal whether sGP-crossreactive antibodies are in fact less protective than GP1,2-specific antibodies. This is particularly important given that passive transfer of anti-EBOV monoclonal antibodies has gained traction recently as a post-exposure therapeutic. If sGP cross-reactivity turns out to be correlated with impaired virus clearance, it would underscore the need to elicit and produce GP1,2-specific antisera or monoclonal antibodies for achieving more effective treatment of EBOV infection. Moreover, our findings also suggest that EBOV vaccines should be tailored to target regions not shared between sGP and GP1,2. This is particularly relevant to recent efforts to develop a broadly-protective vaccine, since these studies have centered around focusing vaccines on conserved epitopes by deleting highly variable regions of GP1,2 such as the mucin domain [24], [43], [47]. Because sGP actually corresponds to the most highly conserved region of GP1, antibodies elicited by these constructs may be cross-reactive with sGP and therefore susceptible to sGP-mediated subversion. Candidate pan-filovirus vaccines may need to be focused on regions of GP1,2 that are both highly conserved and unshared with sGP, such as the membrane-proximal GP2 subunit. It will also be of great interest for EBOV vaccinology to determine whether antigenic subversion correlates with successes and failures of vaccines to protect animals against lethal challenge. It may be critical for an EBOV vaccine to elicit a long lasting immune response with high enough antibody titers so the host can clear the virus before it is able to replicate and effect antigenic subversion. This possibility is consistent with nonhuman primate lethal challenge experiments, in which survival was most closely correlated with maintenance of anti-GP1,2 antibody titers above a threshold level, while lower antibody titers only delayed the time to death [48]. Further, while much of EBOV vaccinology has focused on eliciting protective antibodies against the membrane-bound glycoprotein, a robust T-cell response may also improve vaccine efficacy. Immunization of nonhuman primates with a low dose of GP and nucleoprotein (NP)-expressing recombinant adenoviruses was demonstrated to elicit robust antibody and T-cell responses and confer protection against lethal challenge [49]. More importantly, EBOV-specific T-cells were shown to reduce the threshold of anti-GP1,2 antibodies needed for protection. Recombinant vectors expressing CTL epitopes have been demonstrated to confer protection to lethal EBOV challenge in mice, and GP-specific as well as nucleoprotein (NP)-specific CD8 T-cells can control infection even when adoptively transferred to otherwise naïve animals [50], [51]. These studies suggest that a robust T-cell response may reduce the threshold of antibodies needed for rapid virus clearance. It is noteworthy that although the expression of sGP is conserved in Ebola viruses, sGP is not produced by Marburg virus (MARV), another member of the filoviridae. There are other instances where related viruses often diverge in the mechanisms they employ to survive in their respective hosts. For example, Sendai virus (SeV), a paramyxovirus that causes severe respiratory tract infections in rodents, expresses a V protein via RNA editing of the P gene. V is necessary for in vivo survival and pathogenesis of SeV, though V-deficient SeV show no defect in replication in vitro [52]. However, the closely related human parainfluenza virus type 1 (HPIV-1) does not express V, even though its P gene displays a high degree of homology to SeV P, and HPIV-1 causes similar disease in humans as SeV causes in rodents [53]. Similarly, while secretion of GP has not been observed in MARV, it has likely evolved alternative strategies to survive within its host. While the precise relevance of antigenic subversion to Ebola vaccinology remains to be determined, antigenic subversion represents a novel and elegant solution to the challenge that viruses face of balancing the ability to infect host cells efficiently while evading host immune surveillance. The constraints of a very small genome neccessitate packing a great deal of functionality into a small space, and sGP-mediated subversion represents a mechanism which, along with glycan-dependent steric shielding, and immunodominance of the GP1,2 mucin domain, may help EBOV to survive in its host. Improving our understanding of how these mechanisms work together will eventually open the door to a more rationally designed vaccine. A vaccine directed against highly conserved regions of GP1,2, such as the GP2 subunit, could induce broadly reactive antibodies while also avoiding the potential for sGP-mediated immune subversion. Such a vaccine could protect against multiple strains of EBOV, including strains that have not yet been identified. Materials and Methods Ethics Statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Animal ethics approval for the immunization studies in mice was obtained from the Institutional Animal Care and Use Committee (IACUC) at Emory University. All animal studies were performed under approval from the Institutional Animal Care and Use Committee (IACUC) at Emory University. Female BALB/c mice (8-week old) were purchased from the Jackson Laboratory and housed in the animal facility at the Emory University. Cell Lines and Plasmids 293T cells and HeLa cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM, Mediatech) supplemented with 10% fetal bovine serum (Hyclone, ThermoFisher) and penicillin/streptomycin. All Ebola glycoprotein constructs were based on the Ebola Zaire strain (ZEBOV), Mayinga Subtype (GenBank accession# U23187.1). Editing site mutants were generated in pBlueScript II K/S+ vector through site-directed mutagenesis using the QuickChange XL kit (Stratagene). Constructs were then subcloned pCAGGS mammalian expression vector. Protein expression was carried out by transfecting 90% confluent cells in 6-well plates with 5 µg DNA+12 µL Fugene HD (Roche) per well, as per manufacturer instructions, and detected at 48 h post transfection. Surface expression was detected by surface biotinylation followed by immunoprecipitation with anti-EBOV GP mouse polyclonal antibody, SDS-PAGE, and Avidin-HRP blotting. Cell lysate was harvested in cell lysis buffer and cell culture supernatant was collected, spun down to remove cell debris, and concentrated 10× by a centrifugal concentrator. Cell lysate and concentrated cell culture supernatant were run on SDS-PAGE under denaturing conditions, followed by probing with anti-EBOV GP1,2/sGP rabbit polyclonal antibody. Vaccine Preparation and Immunization Mutant ZEBOV GP plasmids for DNA immunization experiments were prepared using the EndoFree Plasmid Mega Kit (Qiagen) as per manufacturer instructions and redissolved in pure endotoxin-free water at a concentration of 4–6 µg/µL, and purity was verified by restriction analysis and spectrophotometry. For immunization, DNA was diluted in sterile PBS to 0.5 µg/µL and filter sterilized. Female BALB/C mice (Charles River Laboratory) at six mice per group received 50 µg of DNA intramuscularly (25 µg/leg) per immunization. Anesthetized mice were bled retro-orbitally two weeks after each immunization and serum samples were stored at −80°C until use. Recombinant Protein Production and ELISA Production of purified histidine-tagged HA has been described previously [54]. Soluble histidine-tagged GP1,2 and sGP were generated by C-terminal addition of a single 6× histidine tag. Soluble GP1,2 was generated by truncation of the transmembrane domain and cytoplasmic tail. Recombinant vaccinia viruses (rVV) were generated as described elsewhere to synthesize soluble His-tagged GP1,2 (His- GP1,2) and sGP (His-sGP), as well as membrane-bound GP1,2 [55]. For production and purification of His-GP1,2 and His-sGP, rVV-infected cell supernatant was clarified and purified using a PrepEase His Purification Kit (Affymetrix) and purity of recombinant protein was verified by SDS-PAGE followed by Western blot or coomassie stain. Further, purified His-GP1,2 and His-sGP were tested for reactivity to pre-immune sera or sera from unvaccinated mice by ELISA and Western blot, and they were found to be unreactive. For ELISA, flat-bottom Immulon 4-HBX 96-well plates (Thermo) were coated overnight with 0.1 µg/well of His- GP1,2 or His-sGP. A standard curve was generated by coating control wells with known concentrations of mouse IgG. Plates were washed 5× in PBS+Tween (PBST), blocked in PBST+2%BSA, and then incubated in duplicate for two hours with antisera diluted in PBST+2%BSA. Plates were washed again, and incubated with 1∶1000 (pooled anti- IgG subtype) HRP-conjugated goat anti-mouse secondary antibody. After final wash, plates were developed with 3,3′,5,5′-Tetramethylbenzidine (TMB, Thermo) and stopped at 5 minutes with 0.2 M HCl. Plates were read and antibody concentration was calculated using the standard curve. Competition ELISA Competition ELISA was performed by modifying the above protocol. Plates were coated with His- GP1,2. Pooled antisera were diluted in PBST+2%BSA to a concentration corresponding to an OD of 1.0 by anti- GP1,2 ELISA. Diluted antisera were then mixed with decreasing concentrations of purified His-sGP or His- GP1,2 and immediately added to His- GP1,2-coated wells. The ELISA was then developed as described above and competition was calculated as percent of signal compared to no competing antigen. Competition Immunoprecipitation Competition immunoprecipitation was performed by incubating pooled antisera (normalized for anti-GP1,2 titer as determined by ELISA) with 200 ng of purified His- GP1,2 and increasing amounts purified His-sGP at molar ratios of 0.25∶1, 1∶1, 4∶1, and 8∶1 sGP∶GP1,2. Antisera incubated with His-sGP alone, His-GP1,2 alone, or with no GP were used as controls, as well as antisera incubated with GP1,2 in the presence of recombinant influenza HA. Samples were incubated on ice for 20 minutes, followed by addition of protein-G coupled agarose beads (Thermo Scientific) to further incubate at 4°C for an additional two hr with agitation. Samples were then centrifuged and washed three times with with lysis buffer, and then mixed with 6× Laemmli SDS sample buffer with 12% β-mercaptoethanol. The samples were heated at 95°C for 5 minutes and then used for SDS-PAGE followed by Western blot analysis using antibodies gainst both sGP and GP1,2. Affinity of Polyclonal Antisera Apparent affinity of polyclonal antisera was determined by quantitative ELISA using purified IgG from immunized animals. IgG was purified using Melon Gel (Thermo) as per manufacturer instructions and purity of IgG was verified by ELISA and coomassie gel staining. Since quantitative affinity ELISA requires that coating antigen be incubated with increasing dilutions of antibodies until coating antigen becomes saturated, we found that high antibody concentrations can result in signals that exceed the plate reader's range of detection. Thus, we titrated the amount of coating antigen down to 0.05 µg/well to avoid signal saturation. Wells were coated overnight with 0.05 µg of purified His-GP1,2 or His-sGP and after washing and blocking were incubated with purified IgG diluted in PBST+2%BSA, at dilutions ranging from 1∶10 to 1∶1280 (based on original serum volume). ELISAs were developed as described above and the signal converted to nM concentration of IgG by comparison to a standard curve. Apparent Kd's of polyclonal sera were calculated by nonlinear regression analysis using GraphPad Prism. These results were verified manually by analysis of linearized binding curves as detailed elsewhere [33]. Pseudovirus Generation and Neutralizing Assay EBOV-GP pseudotyped HIV was generated as described elsewhere [56]. Briefly, 293T-cells were cotransfected with Env-defective HIV backbone and ZEBOV GP in pCAGGS vector using Fugene HD (Roche). Supernatants were harvested 48 h post-transfection, clarified, and filtered using a 0.45 micron filter. Pseudoviruses were titered by infecting JC53 cells [57], which express β-galactosidase and luciferase under a tat-activated promoter, causing infected cells turning blue with X-Gal staining. Neutralization assays were performed as described elsewhere [56] with minor modifications. Briefly, pseudoviruses were pre-incubated with dilutions of heat-inactivated antisera, and supplemented with heat-inactivated naïve mouse sera (Innovative Research) so that 5% of the total volume was mouse serum. Pseudovirus-antiserum mixtures were then added to 30% confluent JC53 cells and incubated for 48 h. Virus infection and neutralization was measured by luciferase reporter assay, and neutralization was measured by decrease in luciferase expression compared to virus-only controls [57]. We performed a competition neutralization assay by selecting a fixed antisera concentration corresponding to either 50% or 80% neutralizing activity. Diluted antisera were incubated with dilutions of purified His-sGP or with soluble influenza PR8 hemagluttinin (HA) as a control (GenBank Accession# JF690260). Antisera mixtures were then mixed with pseudovirus and the neutralization assay was developed as described above. Interference with neutralization was determined by the percent rescue of infectivity compared to wells with pseudovirus+antisera without competing sGP, as calculated by the formula [(virus+antibody+sGP)−(virus+antibody)]/[(virus alone)−(virus+antibody)]×100. Supporting Information Figure S1 Competition cell surface ELISA. HeLa cells were seeded in a 96-well plate and allowed to grow overnight to 100% confluency. Cells were then infected at an MOI of 5 with a recombinant vaccinia virus that directs infected cells to express membrane-bound EBOV GP1,2. At 24 h post-infection, cells were fixed in 2% paraformaldehyde and washed in PBS. Pooled antisera from mice immunized with sGPEdit (light red), GP-7A (dark red), GP-8A (light blue), or GP1,2Edit (dark blue) were diluted to give roughly equivalent anti-GP1,2 signal. Diluted antiserum was mixed with increasing quantities of purified his-sGP and incubated with fixed GP1,2 expressing cells for two hours to allow sGP to compete with GP1,2 for antibodies. ELISAs were developed as previously described with the exception that detergent-free PBS was used in washing steps. (TIF) Click here for additional data file. Figure S2 Interference with antibody-mediated neutralization by sGP at 50% neutralizing activity. The ability of sGP to interfere with antibody-dependent neutralization was determined identically to Figure 4B, except that the concentration of antisera was fixed to correspond to 50% neutralization. Pooled GP1,2-immunized (blue) and sGP-immunized (red) antisera were co-incubated with increasing dilutions of his-sGP (solid markers) or his-influenza PR8 HA (open markers), and rescue of infectivity was measured as described in methods. (TIF) Click here for additional data file. Figure S3 Expression of GP1,2 and sGP together. Because antigen expression from DNA vaccines is too low to detect in vivo, we measured expression in cell culture as a proxy for in vivo expression. HeLa cells in 6-well plates were transfected with GP1,2Edit, sGPEdit, and empty pCAGGS vector at the same ratio as used to immunize animals and 5 µg total DNA per well. Expression of sGP and GP1,2 was determined 36 h post-transfection in both cell lysate and culture supernatant by Western blot using a polyclonal rabbit antibody that reacts with both GP isoforms. The volume of cell lysate and supernatant analyzed for each sample was proportional to the total amount of lysate and supernatant collected so that the Western blots reflect the relative amounts of total sGP and GP1,2 produced. (TIF) Click here for additional data file. Figure S4 Immunization with lower ratios of sGP∶GP1,2. Female BALB/C mice were immunized IM with 50 µg of total DNA per immunization as in previous immunization experiments and boosted at week 4. The amount of GP1,2Edit was fixed at 12.5 µg, and groups were immunized with 1∶1, 1∶3, and 1∶9 ratios of sGP Edit∶GP1,2 Edit, as well as GP1,2Edit without sGPEdit. Total immunizing DNA was normalized to 50 µg with empty pCAGGS vector. (Top Panel) sGP competition ELISA. Pooled antisera were analyzed from immunized mice at week 6 and the ability of sGP to compete for anti-GP1,2 antibodies was determined by competition ELISA as described in Figure 3B. (Bottom Panel) In Vitro antigen expression. HeLa cells were transfected with GP1,2Edit, sGPEdit, and empty pCAGGS vector at the same ratio as used to immunize animals and 5 µg total DNA per well. Expression of sGP and GP1,2 was determined 36 h post-transfection as describe in Figure S3. Both cell lysate and culture supernatant were analyzed by Western blot using a polyclonal rabbit antibody that reacts with both GP isoforms. (TIF) Click here for additional data file. Figure S5 Interference with antibody-mediated neutralization by sGP at 50% neutralizing activity from GP1,2+sGP antisera. The ability of sGP to interfere with antibody-dependent neutralization was determined identically to Figure 6F, except that the antiserum concentration was fixed to correspond to 50% neutralization. Pooled GP1,2+sGP-immunized antisera were co-incubated with increasing dilutions of sGP (red) or influenza PR8 HA (blue), and rescue of infectivity was measured as described in methods. (TIF) Click here for additional data file.

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Virology. Mutation rate and genotype variation of Ebola virus from Mali case sequences.

T. Hoenen, D Safronetz, A. Groseth … (2015)

The occurrence of Ebola virus (EBOV) in West Africa during 2013-2015 is unprecedented. Early reports suggested that in this outbreak EBOV is mutating twice as fast as previously observed, which indicates the potential for changes in transmissibility and virulence and could render current molecular diagnostics and countermeasures ineffective. We have determined additional full-length sequences from two clusters of imported EBOV infections into Mali, and we show that the nucleotide substitution rate (9.6 × 10(-4) substitutions per site per year) is consistent with rates observed in Central African outbreaks. In addition, overall variation among all genotypes observed remains low. Thus, our data indicate that EBOV is not undergoing rapid evolution in humans during the current outbreak. This finding has important implications for outbreak response and public health decisions and should alleviate several previously raised concerns.

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Journal ID (nlm-journal-id): 0410462

Journal ID (pubmed-jr-id): 6011

Journal ID (nlm-ta): Nature

Journal ID (iso-abbrev): Nature

Title: Nature

ISSN (Print): 0028-0836

ISSN (Electronic): 1476-4687

Publication date Nihms-submitted: 4 April 2015

Publication date (Electronic): 08 April 2015

Publication date (Print): 30 April 2015

Publication date PMC-release: 02 November 2015

Volume: 520

Issue: 7549

Pages: 688-691

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[1 ]Galveston National Laboratory, University of Texas Medical Branch, Galveston, TX, USA.

[2 ]Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA.

[3 ]Department of Virology and Vaccine Vectors, Profectus BioSciences, Inc., Tarrytown, NY, USA

[4 ]Department of Immunology, Profectus BioSciences, Inc., Tarrytown, NY, USA

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[† ]To whom correspondence should be addressed. twgeisbe@ 123456utmb.edu

Article

Manuscript ID: NIHMS677263

DOI: 10.1038/nature14428

PMC ID: 4629916

PubMed ID: 25853476

SO-VID: 4471ac0d-0ae7-4cdb-b5fa-1d8688ac84e2

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Virology. Mutation rate and genotype variation of Ebola virus from Mali case sequences.

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