Bayesian inference for prediction of survival probability in prime-boost vaccination regimes.

We focus on Bayesian inference for survival probabilities in a prime-boost vaccination regime in the development of an Ebola vaccine. We are interested in the heterologous prime-boost regimen (unmatched vaccine deliverys using the same antigen) due to its demonstrated durable immunity, well-tolerated safety profile, and suitability as a population vaccination strategy. Our research is motivated by the need to estimate the survival probability given the administered dosage. To do so, we establish two key relationships. Firstly, we model the connection between the designed dose concentration and the induced antibody count using a Bayesian response surface model. Secondly, we model the association between the antibody count and the probability of survival when experimental subjects are exposed to the Ebola virus in a controlled setting using a Bayesian probability of survival model. Finally, we employ a combination of the two models with dose concentration as the predictor of the survival probability for a future vaccinated population. We implement our two-level Bayesian model in Stan, and illustrate its use with simulated and real-world data. Performance of this model is evaluated via simulation. Our work offers a new application of drug synergy models to examine prime-boost vaccine efficacy, and does so using a hierarchical Bayesian framework that allows us to use dose concentration to predict survival probability.

Dynamics of the Humoral Immune Response to a Prime-Boost Ebola Vaccine: Quantification and Sources of Variation.

The Ebola vaccine based on Ad26.ZEBOV/MVA-BN-Filo prime-boost regimens is being evaluated in multiple clinical trials. The long-term immune response to the vaccine is unknown, including factors associated with the response and variability around the response. We analyzed data from three phase 1 trials performed by the EBOVAC1 Consortium in four countries: the United Kingdom, Kenya, Tanzania, and Uganda. Participants were randomized into four groups based on the interval between prime and boost immunizations (28 or 56 days) and the sequence in which Ad26.ZEBOV and MVA-BN-Filo were administered. Consecutive enzyme-linked immunosorbent assay (ELISA) measurements of the IgG binding antibody concentrations against the Kikwit glycoprotein (GP) were available for 177 participants to assess the humoral immune response up to 1 year postprime. Using a mathematical model for the dynamics of the humoral response, from 7 days after the boost immunization up to 1 year after the prime immunization, we estimated the durability of the antibody response and the influence of different factors on the dynamics of the humoral response. Ordinary differential equations (ODEs) described the dynamics of antibody response and two populations of antibody-secreting cells (ASCs), short-lived (SL) and long-lived (LL). Parameters of the ODEs were estimated using a population approach. We estimated that half of the LL ASCs could persist for at least 5 years. The vaccine regimen significantly affected the SL ASCs and the antibody peak but not the long-term response. The LL ASC compartment dynamics differed significantly by geographic regions analyzed, with a higher long-term antibody persistence in European subjects. These differences could not be explained by the observed differences in cellular immune response.IMPORTANCE With no available licensed vaccines or therapies, the West African Ebola virus disease epidemic of 2014 to 2016 caused 11,310 deaths. Following this outbreak, the development of vaccines has been accelerated. Combining different vector-based vaccines as heterologous regimens could induce a durable immune response, assessed through antibody concentrations. Based on data from phase 1 trials in East Africa and Europe, the dynamics of the humoral immune response from 7 days after the boost immunization onwards were modeled to estimate the durability of the response and understand its variability. Antibody production is maintained by a population of long-lived cells. Estimation suggests that half of these cells can persist for at least 5 years in humans. Differences in prime-boost vaccine regimens affect only the short-term immune response. Geographical differences in long-lived cell dynamics were inferred, with higher long-term antibody concentrations induced in European participants.

Safety and Immunogenicity of a Heterologous Prime-Boost Ebola Virus Vaccine Regimen in Healthy Adults in the United Kingdom and Senegal.

BACKGROUND: The 2014 West African outbreak of Ebola virus disease highlighted the urgent need to develop an effective Ebola vaccine. METHODS: We undertook 2 phase 1 studies assessing safety and immunogenicity of the viral vector modified vaccinia Ankara virus vectored Ebola Zaire vaccine (MVA-EBO-Z), manufactured rapidly on a new duck cell line either alone or in a heterologous prime-boost regimen with recombinant chimpanzee adenovirus type 3 vectored Ebola Zaire vaccine (ChAd3-EBO-Z) followed by MVA-EBO-Z. Adult volunteers in the United Kingdom (n = 38) and Senegal (n = 40) were vaccinated and an accelerated 1-week prime-boost regimen was assessed in Senegal. Safety was assessed by active and passive collection of local and systemic adverse events. RESULTS: The standard and accelerated heterologous prime-boost regimens were well-tolerated and elicited potent cellular and humoral immunogenicity in the United Kingdom and Senegal, but vaccine-induced antibody responses were significantly lower in Senegal. Cellular immune responses measured by flow cytometry were significantly greater in African vaccinees receiving ChAd3 and MVA vaccines in the same rather than the contralateral limb. CONCLUSIONS: MVA biomanufactured on an immortalized duck cell line shows potential for very large-scale manufacturing with lower cost of goods. This first trial of MVA-EBO-Z in humans encourages further testing in phase 2 studies, with the 1-week prime-boost interval regimen appearing to be particularly suitable for outbreak control. CLINICAL TRIALS REGISTRATION: NCT02451891; NCT02485912.

Antibody quality and protection from lethal Ebola virus challenge in nonhuman primates immunized with rabies virus based bivalent vaccine.

We have previously described the generation of a novel Ebola virus (EBOV) vaccine platform based on (a) replication-competent rabies virus (RABV), (b) replication-deficient RABV, or (c) chemically inactivated RABV expressing EBOV glycoprotein (GP). Mouse studies demonstrated safety, immunogenicity, and protective efficacy of these live or inactivated RABV/EBOV vaccines. Here, we evaluated these vaccines in nonhuman primates. Our results indicate that all three vaccines do induce potent immune responses against both RABV and EBOV, while the protection of immunized animals against EBOV was largely dependent on the quality of humoral immune response against EBOV GP. We also determined if the induced antibodies against EBOV GP differ in their target, affinity, or the isotype. Our results show that IgG1-biased humoral responses as well as high levels of GP-specific antibodies were beneficial for the control of EBOV infection after immunization. These results further support the concept that a successful EBOV vaccine needs to induce strong antibodies against EBOV. We also showed that a dual vaccine against RABV and filoviruses is achievable; therefore addressing concerns for the marketability of this urgently needed vaccine.

Antigenic subversion: a novel mechanism of host immune evasion by Ebola virus.

In addition to its surface glycoprotein (GP(1,2)), Ebola virus (EBOV) directs the production of large quantities of a truncated glycoprotein isoform (sGP) that is secreted into the extracellular space. The generation of secreted antigens has been studied in several viruses and suggested as a mechanism of host immune evasion through absorption of antibodies and interference with antibody-mediated clearance. However such a role has not been conclusively determined for the Ebola virus sGP. In this study, we immunized mice with DNA constructs expressing GP(1,2) and/or sGP, and demonstrate that sGP can efficiently compete for anti-GP(12) antibodies, but only from mice that have been immunized by sGP. We term this phenomenon "antigenic subversion", and propose a model whereby sGP redirects the host antibody response to focus on epitopes which it shares with membrane-bound GP(1,2), thereby allowing it to absorb anti-GP(1,2) antibodies. Unexpectedly, we found that sGP can also subvert a previously immunized host's anti-GP(1,2) response resulting in strong cross-reactivity with sGP. This finding is particularly relevant to EBOV vaccinology since it underscores the importance of eliciting robust immunity that is sufficient to rapidly clear an infection before antigenic subversion can occur. Antigenic subversion represents a novel virus escape strategy that likely helps EBOV evade host immunity, and may represent an important obstacle to EBOV vaccine design.

Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses.

Vaccines and therapies are urgently needed to address public health needs stemming from emerging pathogens and biological threat agents such as the filoviruses Ebola virus (EBOV) and Marburg virus (MARV). Here, we developed replication-competent vaccines against EBOV and MARV based on attenuated recombinant vesicular stomatitis virus vectors expressing either the EBOV glycoprotein or MARV glycoprotein. A single intramuscular injection of the EBOV or MARV vaccine elicited completely protective immune responses in nonhuman primates against lethal EBOV or MARV challenges. Notably, vaccine vector shedding was not detectable in the monkeys and none of the animals developed fever or other symptoms of illness associated with vaccination. The EBOV vaccine induced humoral and apparent cellular immune responses in all vaccinated monkeys, whereas the MARV vaccine induced a stronger humoral than cellular immune response. No evidence of EBOV or MARV replication was detected in any of the protected animals after challenge. Our data suggest that these vaccine candidates are safe and highly efficacious in a relevant animal model.

Recombinant Protein Filovirus Vaccines Protect Cynomolgus Macaques From Ebola, Sudan, and Marburg Viruses.

Ebola (EBOV), Marburg (MARV) and Sudan (SUDV) viruses are the three filoviruses which have caused the most fatalities in humans. Transmission from animals into the human population typically causes outbreaks of limited scale in endemic regions. In contrast, the 2013-16 outbreak in several West African countries claimed more than 11,000 lives revealing the true epidemic potential of filoviruses. This is further emphasized by the difficulty seen with controlling the 2018-2020 outbreak of EBOV in the Democratic Republic of Congo (DRC), despite the availability of two emergency use-approved vaccines and several experimental therapeutics targeting EBOV. Moreover, there are currently no vaccine options to protect against the other epidemic filoviruses. Protection of a monovalent EBOV vaccine against other filoviruses has never been demonstrated in primate challenge studies substantiating a significant void in capability should a MARV or SUDV outbreak of similar magnitude occur. Herein we show progress on developing vaccines based on recombinant filovirus glycoproteins (GP) from EBOV, MARV and SUDV produced using the Drosophila S2 platform. The highly purified recombinant subunit vaccines formulated with CoVaccine HT™ adjuvant have not caused any safety concerns (no adverse reactions or clinical chemistry abnormalities) in preclinical testing. Candidate formulations elicit potent immune responses in mice, guinea pigs and non-human primates (NHPs) and consistently produce high antigen-specific IgG titers. Three doses of an EBOV candidate vaccine elicit full protection against lethal EBOV infection in the cynomolgus challenge model while one of four animals infected after only two doses showed delayed onset of Ebola Virus Disease (EVD) and eventually succumbed to infection while the other three animals survived challenge. The monovalent MARV or SUDV vaccine candidates completely protected cynomolgus macaques from infection with lethal doses of MARV or SUDV. It was further demonstrated that combinations of MARV or SUDV with the EBOV vaccine can be formulated yielding bivalent vaccines retaining full efficacy. The recombinant subunit vaccine platform should therefore allow the development of a safe and efficacious multivalent vaccine candidate for protection against Ebola, Marburg and Sudan Virus Disease.

Vesicular stomatitis virus-based ebola vaccine is well-tolerated and protects immunocompromised nonhuman primates.

Ebola virus (EBOV) is a significant human pathogen that presents a public health concern as an emerging/re-emerging virus and as a potential biological weapon. Substantial progress has been made over the last decade in developing candidate preventive vaccines that can protect nonhuman primates against EBOV. Among these prospects, a vaccine based on recombinant vesicular stomatitis virus (VSV) is particularly robust, as it can also confer protection when administered as a postexposure treatment. A concern that has been raised regarding the replication-competent VSV vectors that express EBOV glycoproteins is how these vectors would be tolerated by individuals with altered or compromised immune systems such as patients infected with HIV. This is especially important as all EBOV outbreaks to date have occurred in areas of Central and Western Africa with high HIV incidence rates in the population. In order to address this concern, we evaluated the safety of the recombinant VSV vector expressing the Zaire ebolavirus glycoprotein (VSVDeltaG/ZEBOVGP) in six rhesus macaques infected with simian-human immunodeficiency virus (SHIV). All six animals showed no evidence of illness associated with the VSVDeltaG/ZEBOVGP vaccine, suggesting that this vaccine may be safe in immunocompromised populations. While one goal of the study was to evaluate the safety of the candidate vaccine platform, it was also of interest to determine if altered immune status would affect vaccine efficacy. The vaccine protected 4 of 6 SHIV-infected macaques from death following ZEBOV challenge. Evaluation of CD4+ T cells in all animals showed that the animals that succumbed to lethal ZEBOV challenge had the lowest CD4+ counts, suggesting that CD4+ T cells may play a role in mediating protection against ZEBOV.

Stability and suitability for storage and distribution of Ad26.ZEBOV/MVA-BN®-Filo heterologous prime-boost Ebola vaccine.

The stability profile of a vaccine has important implications for storage, cold chain management and field deployment. The heterologous prime-boost Janssen Ebola vaccine regimen demonstrated an acceptable safety profile and durability of Ebola-specific immune responses in Phase I studies in healthy adults. Potency (infectious titre) of both components of the Ad26.ZEBOV/MVA-BN-Filo regimen were assessed using qPCR-based potency assay and flow cytometry during real-time and accelerated stability studies, conducted between -80 °C and 25 °C. Additionally, vaccine potency was assessed following agitation, temperature cycling, freeze-thawing and while in the injection system. Ad26.ZEBOV remained stable for 24 months when frozen and at 2-8 °C; MVA-BN-Filo remained stable for 24 months frozen and 12 months at 2-8 °C. Potency of both vaccines was maintained during temperature cycling, agitation and freeze-thawing. When exposed to high temperatures (up to 40 °C) in a syringe/needle both vaccines remained stable for at least 6 h. The vaccines are expected to maintain potency for 36 months when frozen (based on extrapolation of observed stability). The findings of this study indicate that the stability of the Ad26.ZEBOV/MVA-BN-Filo is likely suitable for field deployment in regions at risk of Ebola outbreaks, where cold chain maintenance is challenging owing to infrastructure and resource limitations.

Evaluation of Ebola Virus Countermeasures in Guinea Pigs.

Ebola virus (EBOV) pathology in humans remains incompletely understood; therefore, a number of rodent and nonhuman primate (NHP) models have been established to study the disease caused by this virus. While the macaque model most accurately recapitulates human disease, rodent models, which display only certain aspects of human disease but are more cost-effective, are widely used for initial screens during EBOV countermeasure development. In particular, mice and guinea pigs were among the first species used for the efficacy testing of EBOV vaccines and therapeutics. While mice have low predictive value, guinea pigs have proven to be a more reliable predictor for the evaluation of countermeasures in NHPs. In addition, guinea pigs are larger in size compared to mice, allowing for more frequent collection of blood samples at larger volumes. However, guinea pigs have the disadvantage that there is only a limited pool of immunological tools available to characterize host responses to vaccination, treatment and infection. In this chapter, the efficacy testing of an EBOV vaccine and a therapeutic in the guinea pig model are described.

Evaluation of Medical Countermeasures Against Ebolaviruses in Nonhuman Primate Models.

Several ebolavirus species, with varying lethality rates, have caused sporadic outbreaks in Africa resulting in human disease. Ebolaviruses also have the potential for use as biological weapons. Currently, there are no licensed vaccines or therapeutics to respond to outbreaks or deliberate misuse of ebolaviruses. Vaccine or therapeutic efficacy testing of medical countermeasures against ebolaviruses requires an animal model of disease; in vitro testing in cell culture cannot reproduce the complicated balance between host-pathogen interactions required for the ultimate licensure of a countermeasure. Depending on the target of the countermeasure, demonstration of efficacy in the nonhuman primate ebolavirus disease models will most likely be required before licensure. Here, we describe the selection and use of nonhuman primates for vaccine and therapeutic studies against ebolaviruses.

Assessing Antiviral Countermeasures Using Mouse Models of Ebolavirus Infection.

Mouse models of Ebola virus (EBOV) have demonstrated their utility as important tools for screening the efficacy of candidate therapeutics and vaccines. In this chapter we explain the various mouse models that utilize either wild-type or mouse-adapted EBOV variants.

Ebola Vaccination Using a DNA Vaccine Coated on PLGA-PLL/γPGA Nanoparticles Administered Using a Microneedle Patch.

Ebola DNA vaccine is incorporated into PLGA-PLL/γPGA nanoparticles and administered to skin using a microneedle (MN) patch. The nanoparticle delivery system increases vaccine thermostability and immunogenicity compared to free vaccine. Vaccination by MN patch produces stronger immune responses than intramuscular administration.

[New promising treatments of Ebola virus disease are underway]. / Nye lovende behandlinger mod ebola er på vej.

The largest Ebola epidemic ever is about to end. No major breakthrough in terms of specific treatment has been seen, but a number of valuable lessons have been learned, including the potential of intensive supportive care. New products are under development, but clinical trials were initiated late in the epidemic leading to inability to include sufficient numbers. Several large vaccine trials are underway with one vaccine so far showing 100 per cent efficacy. A paradigm shift in accelerated testing of drugs and vaccines during an ongoing epidemic is emerging.

Quantitative serology assays for determination of antibody responses to Ebola virus glycoprotein and matrix protein in nonhuman primates and humans.

The West Africa Ebola virus disease (EVD) outbreak has reached unprecedented magnitude and caused worldwide concerns for the spread of this deadly virus. Recent findings in nonhuman primates (NHPs) demonstrate that antibodies can be protective against EVD. However, the role of antibody response in vaccine-mediated protection is not fully understood. To address these questions quantitative serology assays are needed for measurement of the antibody response to key Ebola virus (EBOV) proteins. Serology enzyme-linked immunosorbent assays (ELISA's), using a reference detection antibody, were developed in order to standardize the quantitation of antibody levels in vaccinated NHPs or in humans exposed to EBOV or immunized with an EBOV vaccine. Critical reagents were generated to support the development of the serology ELISAs. Recombinant EBOV matrix protein (VP40) was expressed in Escherichia coli and purified. Two variants of the glycoprotein (GP), the ectodomain lacking the transmembrane domain (GPΔTM), and an engineered GP lacking the mucin-like domain (GPΔmuc) were expressed and purified from mammalian cell systems. Using these proteins, three ELISA methods were developed and optimized for reproducibility and robustness, including stability testing of critical reagents. The assay was used to determine the antibody response against VP40, GPΔTM, and GPΔmuc in a NHP vaccine study using EBOV virus-like particles (VLP) vaccine expressing GP, VP40 and the nucleoprotein. Additionally, these ELISAs were used to successfully detect antibody responses to VP40, GPΔTM and GPΔmuc in human sera from EBOV infected individuals.

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge.

Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105-106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support the development of Fc fusions of GP as a candidate vaccine for human use.

Aerosolized Ebola vaccine protects primates and elicits lung-resident T cell responses.

Direct delivery of aerosolized vaccines to the respiratory mucosa elicits both systemic and mucosal responses. This vaccine strategy has not been tested for Ebola virus (EBOV) or other hemorrhagic fever viruses. Here, we examined the immunogenicity and protective efficacy of an aerosolized human parainfluenza virus type 3-vectored vaccine that expresses the glycoprotein (GP) of EBOV (HPIV3/EboGP) delivered to the respiratory tract. Rhesus macaques were vaccinated with aerosolized HPIV3/EboGP, liquid HPIV3/EboGP, or an unrelated, intramuscular, Venezuelan equine encephalitis replicon vaccine expressing EBOV GP. Serum and mucosal samples from aerosolized HPIV3/EboGP recipients exhibited high EBOV-specific IgG, IgA, and neutralizing antibody titers, which exceeded or equaled titers observed in liquid recipients. The HPIV3/EboGP vaccine induced an EBOV-specific cellular response that was greatest in the lungs and yielded polyfunctional CD8+ T cells, including a subset that expressed CD103 (αE integrin), and CD4+ T helper cells that were predominately type 1. The magnitude of the CD4+ T cell response was greater in aerosol vaccinees. The HPIV3/EboGP vaccine produced a more robust cell-mediated and humoral immune response than the systemic replicon vaccine. Moreover, 1 aerosol HPIV3/EboGP dose conferred 100% protection to macaques exposed to EBOV. Aerosol vaccination represents a useful and feasible vaccination mode that can be implemented with ease in a filovirus disease outbreak situation.