Periodic Reporting for period 1 - ENDFLU (Evaluation of ratioNally Designed Influenza vaccines)
Reporting period: 2020-08-01 to 2022-01-31
ENDFLU will develop three complementary next-generation influenza vaccination strategies for the world, fueled by the unique combined expertise of Indian and European partners in rational vaccine design and development:
• Create a repository of pre-pandemic GMP-compliant MVA-based vaccine seeds, each encoding an influenza hemagglutinin (HA), based on European partners demonstration of clinical safety and cross-clade immunogenicity of MVA-H5.
• Optimize and select protein- and MVA-based constructs developed by Indian and European ENDFLU partners, resulting in a rationally combined vaccine formulation that induces broadly protective humoral and cell-mediated immunity against all influenza manifestations. This will be advanced in a Phase I clinical trial and Controlled Human Infection Model (CHIM) study.
• Pre-clinically advance other promising protein- and MVA-based constructs toward future development beyond ENDFLU. Constructs to elicit humoral immunity build on rationally-designed and optimized conserved epitopes of influenza surface proteins. MVA-based constructs to elicit cell-mediated immunity will encode conserved internal protein T cell epitopes. The best candidates will be selected based on biochemical, biophysical, functional and manufacturing criteria, and immunogenicity and protective efficacy in mice and ferrets. The selected protein-based construct will enter a Part A Phase I clinical trial in India assessing safety, immunogenicity and optimal dose, to be combined with the selected MVA- based construct. This protein-MVA combination will enter a Part B Phase I clinical trial in the EU, assessing safety and immunogenicity, followed by the CHIM study. A dissemination and exploitation plan, and collaboration with key scientific advocacy organizations will facilitate the adoption of ENDFLU achievements to combat influenza worldwide.
• Create a repository of pre-pandemic GMP-compliant MVA-based vaccine seeds, each encoding an influenza hemagglutinin (HA), based on European partners demonstration of clinical safety and cross-clade immunogenicity of MVA-H5.
• Optimize and select protein- and MVA-based constructs developed by Indian and European ENDFLU partners, resulting in a rationally combined vaccine formulation that induces broadly protective humoral and cell-mediated immunity against all influenza manifestations. This will be advanced in a Phase I clinical trial and Controlled Human Infection Model (CHIM) study.
• Pre-clinically advance other promising protein- and MVA-based constructs toward future development beyond ENDFLU. Constructs to elicit humoral immunity build on rationally-designed and optimized conserved epitopes of influenza surface proteins. MVA-based constructs to elicit cell-mediated immunity will encode conserved internal protein T cell epitopes. The best candidates will be selected based on biochemical, biophysical, functional and manufacturing criteria, and immunogenicity and protective efficacy in mice and ferrets. The selected protein-based construct will enter a Part A Phase I clinical trial in India assessing safety, immunogenicity and optimal dose, to be combined with the selected MVA- based construct. This protein-MVA combination will enter a Part B Phase I clinical trial in the EU, assessing safety and immunogenicity, followed by the CHIM study. A dissemination and exploitation plan, and collaboration with key scientific advocacy organizations will facilitate the adoption of ENDFLU achievements to combat influenza worldwide.
H2 and H9 synthetic target gene sequences have been designed and cloned into MVA vector plasmids. The generation of recombinant MVA with inserted HA gene sequences from selected influenza strains is ongoing. Overall, H2, H5, H7, H9, H10 gene sequences were prioritized to reach the objective of establishing an optimized repository of pre-GMP MVA-ENDFLU vector vaccines from influenza A viruses with pandemic potential.
The following humoral assays have been standardized and validated: hemagglutination inhibition, microneutralization assay, ELISA based on the expression of HA ectodomains, ELLA, ELISA based on expression of HA stem, ELISA based on expression of NA and ELISA based micro-neutralization (MNT). The following cellular assays have been standardized and validated: detection of cytokines by real time polymerase chain reaction (RT-PCR), immunohistochemical detection of influenza antigen in paraffin-fixed ferret tissue sections, intracellular cytokine staining, detection of ferret cytokines by IFN-γ, quantification of T-cell populations in the influenza virus-infected ferret tissue sections.
Protein-based constructs targeting humoral immune responses have been engineered, expressed, purified, and characterized in vitro. These include soluble HA heads and ectofusion constructs, nanoparticle-displayed HA stem constructs, NA constructs, and multivalent constructs. Advanced constructs have demonstrated good antigenicity and stability. Other promising constructs will be further investigated. The production and purification methods of some advanced constructs have been scaled up to 10 litres in bioreactors. The expression of a mini polyepitope sequence is antigenic and leads to epitope specific T cell activation, thereby targeting cellular immune responses against conserved epitopes. Artificial genes encoding the polyepitope have been synthesized, and two sequences have been successfully cloned into the MVA shuttle vector. Another two are in progress. These four MVA-polyepitope constructs will be evaluated in HLA transgenic mice.
Advanced HA-derived constructs were shown to confer full protection against influenza virus infection in mice, including greater breadth of HI titers and protection than a commercially available inactivated influenza vaccine. NA constructs were shown to be immunogenic in mice and elicit protection against homologous challenge. Heterologous challenge studies are ongoing.
The following humoral assays have been standardized and validated: hemagglutination inhibition, microneutralization assay, ELISA based on the expression of HA ectodomains, ELLA, ELISA based on expression of HA stem, ELISA based on expression of NA and ELISA based micro-neutralization (MNT). The following cellular assays have been standardized and validated: detection of cytokines by real time polymerase chain reaction (RT-PCR), immunohistochemical detection of influenza antigen in paraffin-fixed ferret tissue sections, intracellular cytokine staining, detection of ferret cytokines by IFN-γ, quantification of T-cell populations in the influenza virus-infected ferret tissue sections.
Protein-based constructs targeting humoral immune responses have been engineered, expressed, purified, and characterized in vitro. These include soluble HA heads and ectofusion constructs, nanoparticle-displayed HA stem constructs, NA constructs, and multivalent constructs. Advanced constructs have demonstrated good antigenicity and stability. Other promising constructs will be further investigated. The production and purification methods of some advanced constructs have been scaled up to 10 litres in bioreactors. The expression of a mini polyepitope sequence is antigenic and leads to epitope specific T cell activation, thereby targeting cellular immune responses against conserved epitopes. Artificial genes encoding the polyepitope have been synthesized, and two sequences have been successfully cloned into the MVA shuttle vector. Another two are in progress. These four MVA-polyepitope constructs will be evaluated in HLA transgenic mice.
Advanced HA-derived constructs were shown to confer full protection against influenza virus infection in mice, including greater breadth of HI titers and protection than a commercially available inactivated influenza vaccine. NA constructs were shown to be immunogenic in mice and elicit protection against homologous challenge. Heterologous challenge studies are ongoing.
ENDFLU's repository of (pandemic) seed vaccine candidates will be established based on the expression of influenza hemagglutinin (HA) by the Modified Vaccinia Ankara (MVA) vector. This is a technologically- and clinically advanced platform, with demonstrated broad intra-subtypic protective efficacy against influenza, elicited by humoral and cell-mediated immunity, both in animal models and in humans. The regulatory path towards their use upon the emergence of a new influenza pandemic will be established, in consultation with industry and regulatory authorities in EU (EMA) and India (CDSCO).
Pre-clinically well-advanced vaccine candidates will be optimized, by leveraging unique European and Indian scientific progress, strong expertise and know-how in the development of next-generation influenza vaccines. These include protein-based constructs based on a range of target influenza proteins and parts thereof as well as MVA constructs with conserved and/or cellular immunity targets. These ENDFLU vaccine candidates and other promising leads will be further pre-clinically advanced throughout the duration of the project. Those reaching rigorous and critical milestones for their eventual production under Good Laboratory Practice (GLP) conditions will be selected to enter GMP production and Phase I clinical studies. One protein-based vaccine candidate and one MVA-delivered vaccine candidate will be selected based on preclinical stability, safety, immunogenicity and protective efficacy data as well as criteria facilitating their rapid, economical, large-scale production and distribution worldwide, including in low- and middle-income countries (LMICs).
The protein-based vaccine candidate will be tested for safety and immunogenicity in a Phase I dose escalation clinical trial in India. The dose that demonstrates the highest safety and immunogenicity profile will subsequently be combined with the MVA-delivered vaccine candidate as a single MVA-protein formulation to assess its safety and immunogenicity in a Phase I clinical trial, and subsequently to validate its protective efficacy in a CHIM study in the EU.
This approach takes advantage of promising influenza and unique vaccine development strategies of the Indian and European partners to ensure the synergistic development of a next generation rationally designed influenza vaccine candidate that will induce broad and potent humoral immunity against seasonal influenza A and B viruses through the protein-based component and robust and cross-reactive cell-mediated immunity through the MVA-based component.
Finally, a detailed exploitation plan, including a clinical development roadmap, will be prepared for continued clinical development of ENDFLU’s broadly protective influenza vaccine beyond the ENDFLU Phase I clinical trial.
Pre-clinically well-advanced vaccine candidates will be optimized, by leveraging unique European and Indian scientific progress, strong expertise and know-how in the development of next-generation influenza vaccines. These include protein-based constructs based on a range of target influenza proteins and parts thereof as well as MVA constructs with conserved and/or cellular immunity targets. These ENDFLU vaccine candidates and other promising leads will be further pre-clinically advanced throughout the duration of the project. Those reaching rigorous and critical milestones for their eventual production under Good Laboratory Practice (GLP) conditions will be selected to enter GMP production and Phase I clinical studies. One protein-based vaccine candidate and one MVA-delivered vaccine candidate will be selected based on preclinical stability, safety, immunogenicity and protective efficacy data as well as criteria facilitating their rapid, economical, large-scale production and distribution worldwide, including in low- and middle-income countries (LMICs).
The protein-based vaccine candidate will be tested for safety and immunogenicity in a Phase I dose escalation clinical trial in India. The dose that demonstrates the highest safety and immunogenicity profile will subsequently be combined with the MVA-delivered vaccine candidate as a single MVA-protein formulation to assess its safety and immunogenicity in a Phase I clinical trial, and subsequently to validate its protective efficacy in a CHIM study in the EU.
This approach takes advantage of promising influenza and unique vaccine development strategies of the Indian and European partners to ensure the synergistic development of a next generation rationally designed influenza vaccine candidate that will induce broad and potent humoral immunity against seasonal influenza A and B viruses through the protein-based component and robust and cross-reactive cell-mediated immunity through the MVA-based component.
Finally, a detailed exploitation plan, including a clinical development roadmap, will be prepared for continued clinical development of ENDFLU’s broadly protective influenza vaccine beyond the ENDFLU Phase I clinical trial.