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

Development of a vaccine based on recombinant subunit proteins to protect humans and animals against filovirus disease

Ebola Virus Disease (EVD) is the most prominent example of filovirus disease and as a zoonotic virus fits the characteristics of a neglected tropical disease. Despite being characterized as a Category A Priority Pathogen by NIH/NIAID over a decade ago, EVD lacked public and private research resources due to the absence of a commercial market. Previously, outbreaks of limited scale linked to transmission from livestock or wild animals into the human population occurred in the 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 recently in 2013-2015 when an Ebola virus (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. As typical in an epidemic with a significant number of infectious patients traveling within and from the endemic area, the disease was also exported outside the outbreak region as has been shown with introductions into Nigeria, Mali, and the United States (amongst other countries averting in-country transmission from imported cases). This demonstrated the threat posed to the global public health systems if spread of Ebola or a related filovirus cannot be contained at its source. We have produced three soluble recombinant filovirus glycoproteins (GP) and the matrix proteins of EBOV (VP24 and VP40) using the Drosophila S2 cell expression system. For each antigen, a specific immunoaffinity chromatography method was developed to allow purification to purity levels \u3e90%. The immunogenicity of recombinant subunits and admixtures formulated with or without clinically relevant adjuvants was subsequently 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 in mice for protection against challenge with mouse-adapted EBOV. All vaccine formulations containing EBOV GP generated protective responses and serum transfer from such animals into naïve mice demonstrated that humoral immunity alone can be fully protective. Furthermore, the transfer of immune splenocytes into naïve mice showed that recombinant GP and VP24 subunits elicit functional T cell responses that lead to protection against live virus challenge. 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 suggest that vaccination with GP+VP40+VP24 and an emulsion-based adjuvant consistently produces high anti-EBOV IgG and virus neutralizing titers. This 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. Current efforts in our laboratory are focused on defining correlates of protection to allow clinical development of a monovalent vaccine candidate for protection against EVD and further formulation optimization towards a trivalent, recombinant subunit vaccine with protective efficacy against EBOV, Sudan ebolavirus (SUDV) and Marburgvirus (MARV) infection

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 study of common Mendelian disease carriers across ageing British cohorts: meta-analyses reveal heterozygosity for alpha 1-antitrypsin deficiency increases respiratory capacity and height

Background: Several recessive Mendelian disorders are common in Europeans, including cystic fibrosis (CFTR), medium-chain-acyl-Co-A-dehydrogenase deficiency (ACADM), phenylketonuria (PAH) and alpha 1-antitrypsin deficiency (SERPINA1). Methods: In a multicohort study of > 19 000 older individuals, we investigated the relevant phenotypes in heterozygotes for these genes: lung function (forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC)) for CFTR and SERPINA1; cognitive measures for ACADM and PAH; and physical capability for ACADM, PAH and SERPINA1. Results: Findings were mostly negative but lung function in SERPINA1 ( protease inhibitor (PI) Z allele, rs28929474) showed enhanced FEV1 and FVC (0.13 zscore increase in FEV1 (p=1.7×10-5) and 0.16 z-score increase in FVC (p=5.2×10-8)) in PI-MZ individuals. Height adjustment (a known, strong correlate of FEV1 and FVC) revealed strong positive height associations of the Z allele (1.50 cm increase in height (p=3.6×10-10)). Conclusions: The PI-MZ rare (2%) SNP effect is nearly four times greater than the 'top' common height SNP in HMGA2. However, height only partially attenuates the SERPINA1-FEV1 or FVC association (around 50%) and vice versa. Height SNP variants have recently been shown to be positively selected collectively in North versus South Europeans, while the Z allele high frequency is localised to North Europe. Although PI-ZZ is clinically disadvantageous to lung function, PI-MZ increases both height and respiratory function; potentially a balanced polymorphism. Partial blockade of PI could conceivably form part of a future polytherapeutic approach in very short children. The notion that elastase inhibition should benefit patients with chronic obstructive pulmonary disease may also merit reevaluation. PI is already a therapeutic target: our findings invite a reconsideration of the optimum level in respiratory care and novel pathway potential for development of agents for the management of growth disorders

Effects of Alpha Interferon Treatment on Intrinsic Anti-HIV-1 Immunity In Vivo

Alpha interferon (IFN-α) suppresses human immunodeficiency virus type 1 (HIV-1) replication in vitro by inducing cell-intrinsic retroviral restriction mechanisms. We investigated the effects of IFN-α/ribavirin (IFN-α/riba) treatment on 34 anti-HIV-1 restriction factors in vivo. Expression of several anti-HIV-1 restriction factors was significantly induced by IFN-α/riba in HIV/hepatitis C virus (HCV)-coinfected individuals. Fold induction of cumulative restriction factor expression in CD4+ T cells was significantly correlated with viral load reduction during IFN-α/riba treatment (r2 = 0.649; P < 0.016). Exogenous IFN-α induces supraphysiologic restriction factor expression associated with a pronounced decrease in HIV-1 viremia

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

Assessment of Population-Based HIV RNA Levels in a Rural East African Setting Using a Fingerprick-Based Blood Collection Method

Background. Population-based human immunodeficiency virus type 1 (HIV-1) RNA levels (viral load [VL]) are proposed metrics for antiretroviral therapy (ART) program effectiveness. We estimated population-based HIV RNA levels using a fingerprick-based approach in a rural Ugandan community implementing rapid ART scale-up

Overview of systematic reviews. Effective home support in dementia care: Components and impacts, Stage 1, psychosocial interventions for dementia.

Aim: To synthesise evidence to identify the components of effective psychosocial interventions in dementia care to inform clinical practice, policy and research. Background: With population ageing dementia represents a significant care challenge with 60% of people with dementia living at home. Design: Overview of systematic reviews with narrative summary. Data sources: Electronic searches of published systematic reviews in English using Cochrane Database of Systematic Reviews, DARE, EPPI-Centre, between September 2013 - April 2014. Review methods: Systematic reviews were appraised against Cochrane Collaboration levels of effectiveness. Components of psychosocial interventions were identified with their theoretical rationale. Findings were explored with a Patient, Public and Carer Involvement group. Results: 36 systematic reviews were included. From interventions, 14 components were identified, nine for people with dementia and five for carers, mostly undertaken in nursing/care homes. For people with dementia, there was evidence of effectiveness for cognitive stimulation and cognitive training; but less evidence for sensory stimulation, reminiscence, staff education, behavioural therapy and ADL training. For carers, there was evidence of effectiveness for education and training, psychotherapy and counselling. Conclusion: There was a lack of definitive evidence of effectiveness for most psychosocial interventions. Further studies with stronger methodology or replication of existing studies would strengthen the evidence base. Few interventions were undertaken with people with dementia and their carers living at home. Further work will investigate the extent to which components identified here are present in models of home support for people with dementia and carers and their effectiveness

Dementia Caregiver Burden: A Research Update and Critical Analysis

Purpose of Review: This article provides an updated review of the determinants of caregiver burden and depression, with a focus on care demands and especially the differential effects of various neuropsychiatric symptoms or symptom clusters. Moreover, studies on caregivers for frontotemporal and Lewy body dementias were referred to in order to identify differences and similarities with the mainstream literature based largely on Alzheimer caregivers. Recent Findings: As a group, neuropsychiatric symptoms are most predictive of caregiver burden and depression regardless of dementia diagnosis, but the effects appear to be driven primarily by disruptive behaviors (e.g., agitation, aggression, disinhibition), followed by delusions and mood disturbance. Disruptive behaviors are more disturbing partly because of the adverse impact on the emotional connection between the caregiver and the care-recipient and partly because they exacerbate difficulties in other domains (e.g., caring for activities of daily living). In behavioral variant frontotemporal dementia, not only are these disruptive behaviors more prominent but they are also more disturbing due to the care-recipient’s insensitivity to others’ feelings. In Lewy body dementia, visual hallucinations also appear to be distressing. Summary: The disturbing nature of disruptive behaviors cuts across dementia conditions, but the roles played by symptoms that are unique or particularly serious in a certain condition need to be explored further