Development of a standardized multiplex Filovirus and SARS-CoV2 antibody immunoassay

With the goal of producing multivalent recombinant subunit filovirus and SARS-CoV-2 vaccines, we develop formulations using surface glycoproteins of Ebola, Marburg and Sudan viruses or the Spike protein of the SARS-CoV-2 virus. In determining the potency of our formulations in generating an immune response in mice and non-human primates (NHP), serum antibody titers are used. Instead of using conventional antigen-binding ELISA assays for each antigen, we conduct testing by a custom multiplex immunoassay. This method uses regionally different magnetic beads coupled to purified recombinant antigens which are incubated with serum dilutions to simultaneously determine the antibody titers to the different immunizing antigens. After application of a secondary, fluorescently labeled antibody, values are normally shown as median fluorescent intensity or MFI. By converting the MFI to an actual concentration, samples from different studies can more easily be compared. For this, standard curves using purified antigen-specific immunoglobulin G (IgG) to the three filovirus GP’s or SARS-CoV2 spike protein are established with each assay. Standards were prepared passing high-titered mouse or NHP sera over a protein G column to isolate IgG, then purified further using affinity-chromatography columns with individual filovirus GP’s or SARS-CoV-2 spike protein to select for antigen-specificity. The standards are quantified and curves are generated which will be run with each set of serum samples. Please click Download on the upper right corner to see the full abstract

Preclinical development of filovirus and flavivirus vaccines based on recombinant insect cell expressed subunits

Ebola Virus Disease (EVD) is the most prominent example of filovirus disease but despite being characterized as a Category A Priority Pathogen by NIH/NIAID over a decade ago, it lacked public and private research resources due to the absence of a commercial market. Transmission from wild animals into the human population typically causes outbreaks of limited scale in endemic areas located in the forested regions of Central Africa and the Philippines (for Reston ebolavirus), therefore other public health threats garnered more attention. This changed when a Zaire Ebolavirus (EBOV) outbreak of increasing size in several West African countries started to reveal the true epidemic potential that filovirus infections can have when entering an urban setting in a highly mobile society. Despite significant progress with the clinical development of several EBOV vaccine candidates during and after the West African outbreak, no EBOV specific therapeutics and vaccines have yet received regulatory approval. Additional research is needed in particular on understanding the mechanism of protection and defining immune correlates of protection for Ebola and other filovirus vaccines. For our multivalent filovirus vaccine candidate, we have produced soluble recombinant filovirus glycoproteins (GP) from EBOV, Marburg marburgvirus (MARV) and Sudan ebolavirus (SUDV) using the Drosophila S2 cell expression system. The immunogenicity of highly purified recombinant subunits and admixtures formulated with or without clinically relevant adjuvants was evaluated in mice, guinea pigs and macaques. Strong antigen-specific IgG titers as well as virus neutralizing titers were observed after administering two or three doses of adjuvanted formulations. In mice and non-human primates subunit proteins were also shown to elicit cell mediated immune responses. Analysis of secreted cytokines in batch-cultured, antigen-stimulated splenocytes or PBMC’s demonstrated antigen-induced Th1 and Th2 type responses. Recombinant vaccine candidates were tested successfully for protection in the mouse model of EBOV. Further studies allowed us to demonstrate that both humoral and cell-mediated immunity are elicited and can mediate protection. Additional immunogenicity and efficacy studies in guinea pigs were focused on optimized antigen dosing, antigenic balance and adjuvantation. Multiple formulations consistently produced strong antibody responses and demonstrated 100% protective efficacy in the EBOV guinea pig model. Results from studies in two species of non-human primates demonstrate that vaccination with formulations of recombinant EBOV subunits and an emulsion-based adjuvant consistently produces high anti-EBOV IgG and virus neutralizing titers. Such vaccination prevents viremia subsequent to live virus challenge and protects animals from terminal EBOV disease. These studies suggest that we have defined a viable Ebola virus vaccine candidate based on non-replicating viral subunits. In addition to updates on efficacy testing against EBOV and MARV, we will discuss current formulation optimization efforts in our laboratory including thermostabilization of recombinant subunits as well as defining correlates of protection. These are prerequisites to enable efficient clinical development of a monovalent vaccine candidate for protection against EVD and a multivalent, recombinant subunit vaccine with protective efficacy against EBOV, SUDV and MARV infection. Recently we have also demonstrated the applicability of our vaccine platform for the rapid development of vaccines against emerging diseases with a focus on Zika virus (ZIKV), a flavivirus, where we were able to demonstrate efficacy in mice and cynomolgus macaques within approximately 13 months from designing the synthetic gene for antigen expression. While a completely different disease from EVD, the recent outbreak of ZIKV in the Americas provided a similar challenge as no vaccine development efforts have been conducted prior to 2016 and an increasing body of evidence suggests that rather than causing a typical, mild form of disease as previously reported, ZIKV infections can cause neurological sequelae as well as fetal and infant malformations. These results demonstrate that the insect cell expression system can be used to rapidly and efficiently produce recombinant viral subunits from a variety of pathogenic viruses that are highly immunogenic in multiple animal species and are capable of providing effective vaccine protection against live virus challenge

Development of a thermostable, multivalent filovirus vaccine based on recombinant subunit proteins

Ebola Virus Disease (EVD) is the most prominent example of filovirus disease but despite being characterized as a Category A Priority Pathogen by NIH/NIAID over a decade ago, it lacked public and private research resources due to the absence of a commercial market. Transmission from wild animals into the human population typically causes outbreaks of limited scale in endemic areas located in the forested regions of Central Africa and the Philippines (for Reston ebolavirus). In the past decade, a Zaire Ebolavirus (EBOV) outbreak causing more than 11,000 deaths in several West African countries started to reveal the true epidemic potential that filovirus infections can have when entering an urban setting in a highly mobile society. In addition a persistent outbreak in the Democratic Republic of the Congo has continued since August 2018 despite significant progress with the clinical development of several EBOV vaccine candidates (one of which recently gained regulatory approvals in Europe, the U.S. and several African countries) and the advanced testing of promising EBOV specific therapeutics. Despite this significant progress, additional research is needed in particular on understanding the mechanism of protection and defining immune correlates of protection for Ebola and other filoviruses do develop fast and efficacious strategies for outbreak control as the incidence of outbreaks and total case numbers has significantly increased over the last decadesPlease click Download on the upper right corner to see the full abstract

A Recombinant Subunit Based Zika Virus Vaccine Is Efficacious in Non-human Primates

Zika Virus (ZIKV), a virus with no severe clinical symptoms or sequelae previously associated with human infection, became a public health threat following an epidemic in French Polynesia 2013–2014 that resulted in neurological complications associated with infection. Although no treatment currently exists, several vaccines using different platforms are in clinical development. These include nucleic acid vaccines based on the prM-E protein from the virus and purified formalin-inactivated ZIKV vaccines (ZPIV) which are in Phase 1/2 clinical trials. Using a recombinant subunit platform consisting of antigens produced in Drosophila melanogaster S2 cells, we have previously shown seroconversion and protection against viremia in an immunocompetent mouse model. Here we demonstrate the efficacy of our recombinant subunits in a non-human primate (NHP) viremia model. High neutralizing antibody titers were seen in all protected macaques and passive transfer demonstrated that plasma from these NHPs was sufficient to protect against viremia in mice subsequently infected with ZIKV. Taken together our data demonstrate the immunogenicity and protective efficacy of the recombinant subunit vaccine candidate in NHPs as well as highlight the importance of neutralizing antibodies in protection against ZIKV infection and their potential implication as a correlate of protection

Detection of Sugarcane Yellow Leaf Luteovirus of India

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Ebola Virus Glycoprotein Induces an Innate Immune Response In vivo via TLR4

Ebola virus (EBOV), a member of the Filoviridae family, causes the most severe form of viral hemorrhagic fever. Although no FDA licensed vaccine or treatment against Ebola virus disease (EVD) is currently available, Ebola virus glycoprotein (GP) is the major antigen used in all candidate Ebola vaccines. Recent reports of protection as quickly as within 6 days of administration of the rVSV-based vaccine expressing EBOV GP before robust humoral responses were generated suggests that the innate immune responses elicited early after vaccination may contribute to the protection. However, the innate immune responses induced by EBOV GP in the absence of viral vectors or adjuvants have not been fully characterized in vivo. Our recent studies demonstrated that immunization with highly purified recombinant GP in the absence of adjuvants induced a robust IgG response and partial protection against EBOV infection suggesting that GP alone can induce protective immunity. In this study we investigated the early immune response to purified EBOV GP alone in vitro and in vivo. We show that GP was efficiently internalized by antigen presenting cells and subsequently induced production of key inflammatory cytokines. In vivo, immunization of mice with EBOV GP triggered the production of key Th1 and Th2 innate immune cytokines and chemokines, which directly governed the recruitment of CD11b+ macrophages and CD11c+ dendritic cells to the draining lymph nodes (DLNs). Pre-treatment of mice with a TLR4 antagonist inhibited GP-induced cytokine production and recruitment of immune cells to the DLN. EBOV GP also upregulated the expression of costimulatory molecules in bone marrow derived macrophages suggesting its ability to enhance APC stimulatory capacity, which is critical for the induction of effective antigen-specific adaptive immunity. Collectively, these results provide the first in vivo evidence that early innate immune responses to EBOV GP are mediated via the TLR4 pathway and are able to modulate the innate-adaptive interface. These mechanistic insights into the adjuvant-like property of EBOV GP may help to develop a better understanding of how optimal prophylactic efficacy of EBOV vaccines can be achieved as well as further explore the potential post-exposure use of vaccines to prevent filoviral disease

Maternal Immunization Using a Protein Subunit Vaccine Mediates Passive Immunity against Zaire ebolavirus in a Murine Model

The Ebola virus has caused outbreaks in Central and West Africa, with high rates of morbidity and mortality. Clinical trials of recombinant virally vectored vaccines did not explicitly include pregnant or nursing women, resulting in a gap in knowledge of vaccine-elicited maternal antibody and its potential transfer. The role of maternal antibody in Ebola virus disease and vaccination remains understudied. Here, we demonstrate that a protein subunit vaccine can elicit robust humoral responses in pregnant mice, which are transferred to pups in breastmilk. These findings indicate that an intramuscular protein subunit vaccine may elicit Ebola-specific IgG capable of being transferred across the placenta as well as into the breastmilk. We have previously shown protective efficacy with these vaccines in non-human primates, offering a potential safe and practical alternative to recombinant virally vectored vaccines for pregnant and nursing women in Ebola endemic regions