A Randomized, Blinded, Dose-Ranging Trial of an Ebola Virus Glycoprotein (EBOV GP) Nanoparticle Vaccine with Matrix-M™ Adjuvant in Healthy Adults.

BACKGROUND
Ebola virus (EBOV) epidemics pose a major public health risk. There currently is no licensed human vaccine against EBOV. The safety and immunogenicity of a recombinant EBOV glycoprotein (GP) nanoparticle vaccine formulated with or without Matrix-M™ adjuvant were evaluated to support vaccine development.


METHODS
A phase 1, placebo-controlled, dose-escalation trial was conducted in 230 healthy adults to evaluate 4 EBOV GP antigen doses as single- or 2-dose regimens with or without adjuvant. Safety and immunogenicity were assessed through 1-year post-dosing.


RESULTS
All EBOV GP vaccine formulations were well tolerated. Receipt of 2 doses of EBOV GP with adjuvant showed a rapid increase in anti-EBOV GP IgG titers with peak titers observed on Day 35 representing 498- to 754-fold increases from baseline; no evidence of an antigen dose-response was observed. Serum EBOV-neutralizing and binding antibodies using wild-type ZEBOV or pseudovirion assays were 3- to 9-fold higher among recipients of 2-dose EBOV GP with adjuvant, compared with placebo on Day 35, which persisted through 1 year.


CONCLUSIONS
EBOV GP vaccine with Matrix-M adjuvant is well tolerated and elicits a robust and persistent immune response. These data suggest that further development of this candidate vaccine for prevention of EBOV disease is warranted.


CLINICAL TRIALS REGISTRATION
ClinicalTrials.gov [NCT02370589]; anzctr.org.au [EBOV-H-101].

The Ebola virus (EBOV), belonging to the Filoviridae family of viruses, is a human pathogen that poses an important public health challenge to nations already burdened with multiple health and economic adversities. Four of five identified species of EBOV, which include Zaire, Sudan, Tai Forest, and Bundibugyo, share the ability to quickly spread from person to person and cause serious hemorrhagic disease with a high fatality rate. The largest EBOV outbreak to date, starting in 2013, afflicted almost 30 000 people, almost half of which died [1]. No licensed prophylactic or therapeutic drugs or vaccines currently exist for human use against EBOV. Several experimental candidate vaccines are being tested in various phases of clinical trials, with the most advanced candidates being live-viral vectored products based on vesicular stomatitis virus, adenovirus, or vaccinia virus [2]. One candidate (recombinant vesicular stomatitis virus-Zaire EBOV [rVSV-ZEBOV]) has undergone evaluation of effectiveness in the field in an open-label, clusterrandomized trial in Guinea and showed 100% efficacy, although this result continues to be debated [2,3]. This same candidate is now being deployed to address an ongoing urban outbreak in the Democratic Republic of Congo [4,5]. Otherwise, current strategies for treatment and control of infection are limited to supportive care and patient quarantine, making effective control of an outbreak an extremely difficult task [6].
Vaccines against EBOV offer a promising strategy for infection control. An ideal vaccine would be rapidly immunogenic, granting long-term protection against EBOV, with minimal side effects. The several experimental candidate vaccines against EBOV in clinical trials include an rVSV-based vaccine, adenovirus-based vaccines, and modified vaccinia Ankara-Bavarian Nordic virus (MVA-BN) [3,[5][6][7][8][9][10][11]. Most vaccine candidates have targeted the EBOV envelope glycoprotein (GP), which is expressed on the viral envelope as trimeric spikes that mediate attachment and fusion of the virus to the host cell [12,13]. The GP expressed by most experimental vaccines is derived from the 1976 ZEBOV strain. The only published exception has been an adenovirus type 5-based vaccine that expresses the Guinea variant (also known as the Makona variant) of ZEBOV, responsible for the 2014 outbreak [10]. Although live virusvectored vaccines have utility in ring-vaccination strategies for outbreak containment, their use in broad-based vaccination campaigns targeting durable population-based immunity is confounded by a variety of issues, including cold chain requirements, safety concerns in pregnant women and the immunocompromised, and the potential inability to boost in the face of vector immunity.
To our knowledge, the EBOV GP nanoparticle vaccine candidate (Novavax, Inc., Gaithersburg, MD) is the only other vaccine in clinical trials expressing the full-length 2014 Guinea variant GP (Homo sapiens-wt/SLE/2014/Makona G3798) [14,15]. The baculovirus and insect cell Spodoptera frugiperda (Sf9)expression technology has been used to produce other properly folded virus GPs, such as respiratory syncytial fusion protein, which elicit neutralizing antibody responses in animal models and humans and bind a range of neutralizing antibodies that interact with both linear and conformation-dependent epitopes [16,17]. This expression system was used to rapidly produce Good Manufacturing Practice material ready for clinical testing within 11 weeks of publication of the GP sequence. The EBOV GP antigen has been shown to elicit protective immunity in mouse models [14,15] and to produce high-titer human anti-EBOV GP immunoglobulin G (IgG) in transchromosomic bovines that conferred passive protection on nonhuman primates subjected to lethal EBOV challenge [18,19]. In this study, we present the safety and immunogenicity experience for the EBOV GP nanoparticle vaccine, administered with or without the saponin-based adjuvant Matrix-M (Novavax, Inc.), as evaluated in a first-in-human clinical trial in healthy adults.

Clinical Trial Design and Oversight
This randomized, observer-blind, dose-ranging, placebocontrolled, phase 1 clinical trial was conducted at 3 sites in Australia between February 11, 2015 andApril 19, 2016 (ClinicalTrials.gov [NCT02370589]; anzctr.org.au [EBOV-H-101]). A total of 230 healthy male and female participants, 18 to 50 years of age, were randomized to 1 of 13 treatment groups to evaluate the safety and immunogenicity of placebo or 1 of 4 escalating doses of EBOV GP nanoparticle vaccine (6.5, 13, 25, or 50 µg) given intramuscularly (0.5 mL) as a 1-or 2-dose regimen administered on Days 0 and 21, formulated with or without Matrix-M adjuvant (50 µg) (Supplementary Table S1). Enrollment was performed in 3 stages and supervised by an independent safety monitoring committee, which reviewed safety data from each stage against prespecified holding criteria before enrolling each subsequent stage. No vaccination holding criteria were met at any point. The trial was conducted in accordance with the International Conference on Harmonisation Good Clinical Practice and the Declaration of Helsinki. The protocol and informed consent were reviewed and approved by the Belberry Human Research Ethics Committee. All participants provided written informed consent.

Objectives
The primary objectives were 3-fold: (1) to accumulate a vaccine candidate safety experience based on solicited short-term reactogenicity; 84-day all adverse-event (AE) profile; 1-year medically attended AEs (MAEs), serious AEs (SAEs), and significant new medical condition (SNMC) profiles; and selected pre-and postimmunization clinical laboratory parameters; (2) to determine an adjuvant effect of Matrix-M adjuvant based on serum IgG antibodies to EBOV GP at 35 days postdose 1; and (3) to select the minimal EBOV GP antigen dose that, when administered with Matrix-M adjuvant, elicited an anti-EBOV GP IgG antibody response at Day 35 that was greater than or equal to that of the highest dose of unadjuvanted vaccine. Secondary objectives included evaluation of serum neutralizing antibody titers against EBOV and whole virus binding antibodies.

Test Articles
Recombinant EBOV GP was produced by cloning the fulllength GP gene (H. sapiens-wt/SLE/2014/Makona-G3798 [GenBank AIG96283]) of 2014 Makona EBOV into recombinant baculovirus, expression in Sf9 cells, chromatographic purification as described by Bengtsson et al [14,15], and formulated in 25 mM sodium phosphate buffer, pH 6.0, 150 mM sodium chloride, and 0.01% polysorbate-80. The insect cellexpressed EBOV GP (used as both vaccine antigen and target of antibody binding in the GP enzyme-linked immunosorbent assay [ELISA]) was characterized for the display of both conformational and linear neutralizing epitopes by use of surface plasmon resonance to demonstrate high-affinity binding of 3 well characterized neutralizing and/or protective monoclonal antibodies. KZ52, a human monoclonal antibody that recognizes an epitope at the chalice-like base formed from GP1 attachment subunits and pre-fusion GP2, 13C6, a humanmouse chimeric antibody that binds to a conformational epitope on GP1 and is protective in nonhuman primates (NHP), and 6D8, a human-mouse chimeric antibody that recognizes a linear epitope in the mucin-like domain of GP2 and is both neutralizing and protective in NHPs [12,13,20]. All bound with high affinity to purified EBOV GP (Supplementary Table  S2) and provide evidence that EBOV GP expressed in insect cells is correctly folded and presents multiple neutralizing epitopes. The Matrix-M adjuvant comprises 2 distinct saponin fractions (A and C) purified from the bark of the Quillaja saponaria tree and formulated with egg-derived phosphatidylcholine and synthetic cholesterol to form stable particles of approximately 36 ± 4 nm diameter [14,15]. Matrix-M adjuvant is produced by blending the 2 components in a ratio of 85:15. The EBOV GP antigen was supplied to the clinic at 4 different concentrations and mixed with Matrix-M adjuvant, as required, by trained site staff and according to protocol-specified ratios, immediately before administration. Placebo was sterile isotonic saline for injection.

Safety Assessments
All participants had scheduled visits on Days 0, 7, 21, 28, 35, 84, 182, and 385. Vital signs were collected at all visits through Day 84, and clinical laboratory safety testing was performed on Days 0, 7, 21, and 28. Subject-recorded local (injection site pain, redness, swelling, and bruising) and systemic (fatigue, headache, muscle pain, diarrhea, nausea, joint pain, chills, vomiting, and fever) solicited AEs occurring within 7 days after each dose (ie, short-term reactogenicity) were recorded, as well as all unsolicited AEs through Day 84. In addition, MAEs, SAEs, and SNMCs were sought through Day 385.

Immunogenicity Assessments
The primary immunogenicity endpoint was anti-EBOV GP antibody concentration as determined by ELISA as described previously [14], with modification for detection of human IgG. Human anti-EBOV GP serum from a previously immunized transchromosomal bovine source [18,19] was used as a reference standard, and normal human serum was used as a negative control. Anti-EBOV GP IgG ELISA was performed on sera collected on Days 0, 7, 21, 28, 35, 84, 182, and 385. Secondary endpoints, tested on a random subset of subjects selected before generation of any immunogenicity data, included serum antibody titers against whole inactivated ZEBOV Makona [21] and neutralizing antibodies against wild-type ZEBOV Mayinga [7], both assayed at Philipps University of Marburg (neutralizing titers were assessed under biosafety level 4 conditions). Neutralizing titers were also assayed using VSV pseudovirions encoding luciferase reporter and the GP of ZEBOV (strain Kikwit-95) at the US Army Medical Research Institute of Infectious Diseases (USAMRIID) [3].

Statistical Analysis
The safety population, comprising all subjects who received any test article, was used to summarize participant safety experience overall and by treatment group based on the parameters described above. The numbers and percentages (95% confidence interval [CI]) of participants in each treatment group reporting a given AE term were summarized by treatment, severity, and investigator-assessed relatedness to test article.
The per-protocol (PP) population, comprising subjects who received treatment as randomized and had no major protocol deviations, was the primary analysis population for immunogenicity. Descriptive analyses based on the modified intent-totreat population had no differential impact on the conclusions.
The anti-EBOV GP IgG antibody response was characterized by geometric mean ELISA units ([GMEU] with 95% CIs) by treatment group at baseline and postvaccination on Days 7, 21, 28, 35, 84, 182, and 385, and geometric mean ratio (GMR) of postimmunization GMEU relative to baseline GMEU at the same time points. Secondary endpoints were summarized based on geometric mean titers (GMT) in the various neutralization assay formats. Adjuvant effect was tested using the anti-EBOV GP IgG ELISA and summarized using the GMR and corresponding 95% CI. Given that ratio data such as GMR are typically not normally distributed, a log 10 transformation was performed before statistical analysis. An analysis of covariance repeated-measures model with fixed effects for treatment group and treatment-by-antigen interactions was applied. Modeling was performed using PROC MIXED in SAS. Vaccine group comparisons for an adjuvant effect were assessed using a t test; P < .05 was considered significant.

Trial Participants
A total of 230 participants were randomized to 1 of 13 treatment groups (Supplementary Table S1). Approximately 71% of all participants were female (53% to 100% per treatment group), and the majority of participants self-identified as white (73% to 93%) and non-Hispanic (93% to 100%). Mean height, weight, and age (23.5 to 31.9 years) were similar among treatment groups (Table 1). All subjects received the assigned test article on Day 0, and 87% to 100% (by treatment group) received a second dose on Day 21. Approximately 18% of the participants overall discontinued the trial (Figure 1), most commonly at later time points, but no discontinuations were reported as due to an AE. All randomized participants were included in the safety population. The PP population by treatment group included 53.3% to 100% of participants. Reasons for exclusion from the PP population were primarily out of window or missed serology visits ( Figure 1).

Safety Experience
There were no deaths or discontinuations due to AEs in the trial. The proportion of subjects with short-term reactogenicity, including local and systemic AEs, was clearly increased among recipients of the adjuvanted formulations compared with unadjuvanted or placebo recipients ( Figure 2). This effect was most prominent after the second dose of the adjuvanted vaccine. There was no obvious EBOV GP antigen dose-response in reactogenicity. Common reactogenicity complaints were injection site pain and swelling, headache, fatigue, and myalgia (Supplementary Tables S3 and S4). The majority of solicited reactogenicity events were mild to moderate in severity ( Table 2) and resolved within the 7-day solicitation period. Between 47% and 87% of subjects in the various treatment groups reported at least 1 unsolicited AE during the study, with no apparent association of increased risk with active vaccine, adjuvant, or 2-dose regimen ( Table 2). Adverse events deemed severe were infrequent, occurring in no more than 2 subjects in any active group; investigators assessed only 3 such events, including 1 event in the placebo group, as possibly related to test article, again with no clear association with higher antigen dose or The safety population included all study subjects who provided consent, were randomized, and received at least 1 dose of test article. The safety population was used for all safety analyses. The intent-to-treat (ITT) population included all subjects in the safety population who provided any Ebola virus glycoprotein (EBOV GP) serology data, was the secondary population used for immunogenicity analyses, and was analyzed according to treatment as randomized. The per-protocol (PP) population included all subjects in the safety population who received the assigned doses of test article on Day 0 and Day 21 as randomized, had EBOV GP serology results for at least baseline (Day 0), Day 21, and Day 35, and had no major protocol deviations affecting the primary immunogenicity outcomes as determined by Novavax before database lock and unblinding. The PP population was the primary population for immunogenicity analyses. "Other reasons" for participant withdrawal refer to 2 participants who relocated away from the study site. adjuvant. Medical attendance for AEs occurred in 20.0% to 37.5% of subjects and was evenly distributed across groups (Table 2). Nine SAEs occurred in 7 subjects and involved a range of diagnoses in 6 organ systems (Supplementary Table S5); only 2 occurred within 1 month of a vaccine dose, and those were sequelae of accidental trauma. No other SAEs occurred within 4 months of an active vaccine dose, none were deemed treatment-related, and all resolved within the study period. A review of the clinical laboratory assessments did not suggest any systemic toxicity pertaining to the renal or hepatobiliary systems, or to the bone marrow or circulating formed elements of the blood.

Immune Responses Against Ebola Virus Glycoprotein
Baseline GMEU for anti-EBOV GP IgG were 80 to 131 across all treatment groups and approximated the assay lower limit of quantitation. Peak responses were observed on Day 35 and were 2-to 3-fold over baseline in recipients of 2 doses of unadjuvanted EBOV GP (Figure 3, Supplementary   Severe solicited AEs a n (%)  Severe unsolicited AEs b n (%)    Table S7). Anti-EBOV GP IgG levels remained persistently elevated, especially in the 2-dose adjuvanted vaccine groups, through 385 days (Figure 3, Supplementary Table S6).

Other Measures of Immune Response Against the Ebola Virus
Antibodies were measured using an inactivated whole-virus capture ELISA and by 2 neutralization methods using either infectious wild-type Mayinga EBOV [7] or VSV pseudovirions expressing the EBOV GP [3]. Due to resource constraints on these assays, a random cross-sectional sampling of sera at Day 35 was tested, followed by tests of persistence at Days 182 and 385 on available sera from the same subjects. Mean antibody titers obtained using EBOV whole-virus capture ELISA with Day 35 sera were markedly increased among subjects who received 2 doses of EBOV GP with Matrix-M adjuvant in contrast to subjects who received 1 dose of adjuvanted vaccine, 2 doses of unadjuvanted vaccine, or placebo; and the wild-type EBOV neutralizing data tracked this pattern ( Figure 4, Table  S8). Neutralizing antibody titers obtained using USAMRIID's pseudovirion neutralization assay ([PsVNA] 50%) behaved similarly with Day 35 GMT among 2-dose adjuvanted participants ranging from 1154 to 1736 (Figure 4, Supplementary  Table S8), compared with GMT ~10 among placebo and 2-dose unadjuvanted participants. The relationship between the Novavax EBOV GP ELISA units in sera collected at 35 days with whole-virion ELISA titers, PsVNA50 titers, and wild-type Mayinga neutralizing titers was assessed. All 3 functional virus assays behaved similarly and had a sharp upstroke at 5-10 000 Novavax ELISA units ( Figure 5).

DISCUSSION
Ebola virus poses a major public health threat given its ability to quickly infect people that come in contact with a sick patient and to cause severe hemorrhagic disease characterized by rapid progression to multiple-organ failure, shock, and death in 50% to 90% of cases. A vaccine against EBOV could potentially prevent and/or contain Ebola epidemics and eliminate the high mortality associated with them. Recent experience in both the West African outbreak of 2013-2016 and the Democratic Republic of Congo outbreak in 2018 suggest that a VSV-vectored EBOV vaccine may have been effective, but conclusive evidence for vaccine safety and efficacy has not been established [2,6,22].  We have developed an EBOV GP nanoparticle vaccine, based on the Guinea variant of the ZEBOV isolated from the most widespread EBOV outbreak to date in 2014, using the baculovirus/Sf9-expression technology. In this study, we have described the results from a phase 1 clinical trial evaluating the immunogenicity and safety of 4 different doses of the EBOV GP nanoparticle vaccine administered with or without Matrix-M adjuvant as a single-or double-dose regimen. The vaccine, given at any of the 4 antigen doses tested with or without Matrix-M adjuvant, was generally well tolerated among participants. Among the dose regimens, the 2-dose Matrix-M adjuvant elicited a remarkably high and persistent anti-EBOV GP IgG response and exhibited an antigen dose-sparing response in that responses to the 6.5-, 13-, 25-, and 50-µg antigen doses combined with Matrix-M adjuvant were indistinguishable. A similar dose-sparing effect of saponin adjuvant in naive subjects was previously demonstrated using baculovirus/Sf9-derived influenza A/H7N9 virus-like particles [23]. The functional nature and persistence of the immune response out to 1-year postvaccination was confirmed (1) using an inactivated whole-virus capture ELISA and (2) by 2 neutralization methods using either infectious wild-type Mayinga EBOV or VSV pseudovirions expressing the Kikwit EBOV GP. In murine studies, immunization with EBOV/Makona GP with Matrix-M adjuvant was 100% protective in a lethal viral challenge, whereas only 20% of animals   immunized intramuscularly with the unadjuvanted vaccine survived the challenge [15]. In mice, Matrix-M-adjuvanted vaccine induced a rapid onset of specific IgG and neutralizing antibodies, increased frequency of antigen-specific multifunctional CD4 + and CD8 + T cells, specific T-follicular helper cells, germinal center B cells, and persistence of EBOV GP-specific plasma B cells in the bone marrow, suggesting that multiple arms of the immune system are engaged and may contribute to protection [14].

CONCLUSIONS
Although the breadth of the observed clinical immunogenicity analyses using several antibody assays suggest that this adjuvanted recombinant vaccine has the potential to provide protection in the field, either a field efficacy trial or relevant animal challenge studies are needed to define species-independent correlates of protection and to support regulatory approval under the "Animal Rule" [24]. The USAMRIID PsVNA assay has been used to evaluate neutralizing antibodies in NHP challenge studies and multiple human clinical trials [24]; if neutralizing antibodies measured in this assay were deemed to be predictive of protection, then is reasonable to compare responses from a vaccine currently being used in the field with the responses from Novavax's EBOV GP vaccine. The VSV-ZEBOV vaccine delivered at a single dose of 2 × 10 7 plaque-forming units (PFUs) has been shown to potentially provide protection in a ring vaccination study [3] and is being deployed to contain an EBOV epidemic in the Democratic Republic of the Congo [4]. In phase 1 clinical studies, the VSV-ZEBOV vaccine at the 2 × 10 7 PFU dose elicited PsVNA50 GMT of 441 (95% CI, 236-825) on Day 28 in the United States [5] and slightly lower levels in Africa (GMT = 126; 95% CI, 56-285) [25]. In this study, 2 doses of 6.25 μg of recombinant nanoparticle EBOV GP vaccine plus adjuvant elicited comparatively higher levels of neutralizing antibodies: PsVNA50 GMT on Day 35 was 1736.0 (95% CI, 1145.43-2630.95) and titers remained elevated above baseline through Day 385 ( Figure 4). In addition to a surrogate marker of protection, other important details, such as the percentage of subjects reaching the criteria for "protective" responses, have yet to be defined, but if this could be achieved, Novavax's recombinant nanoparticle EBOV GP vaccine could offer a valuable option for the prevention of EBOV. Taken together, the EBOV GP nanoparticle vaccine administered with Matrix-M adjuvant as a 2-dose regimen appears highly promising and warrants further

Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.