Formulation development and comparability studies with an aluminum-salt adjuvanted SARS-CoV-2 spike ferritin nanoparticle vaccine antigen produced from two different cell lines.

The development of safe and effective second-generation COVID-19 vaccines to improve affordability and storage stability requirements remains a high priority to expand global coverage. In this report, we describe formulation development and comparability studies with a self-assembled SARS-CoV-2 spike ferritin nanoparticle vaccine antigen (called DCFHP), when produced in two different cell lines and formulated with an aluminum-salt adjuvant (Alhydrogel, AH). Varying levels of phosphate buffer altered the extent and strength of antigen-adjuvant interactions, and these formulations were evaluated for their (1) in vivo performance in mice and (2) in vitro stability profiles. Unadjuvanted DCFHP produced minimal immune responses while AH-adjuvanted formulations elicited greatly enhanced pseudovirus neutralization titers independent of ∼100%, ∼40% or ∼10% of the DCFHP antigen adsorbed to AH. These formulations differed, however, in their in vitro stability properties as determined by biophysical studies and a competitive ELISA for measuring ACE2 receptor binding of AH-bound antigen. Interestingly, after one month of 4°C storage, small increases in antigenicity with concomitant decreases in the ability to desorb the antigen from the AH were observed. Finally, we performed a comparability assessment of DCFHP antigen produced in Expi293 and CHO cells, which displayed expected differences in their N-linked oligosaccharide profiles. Despite consisting of different DCFHP glycoforms, these two preparations were highly similar in their key quality attributes including molecular size, structural integrity, conformational stability, binding to ACE2 receptor and mouse immunogenicity profiles. Taken together, these studies support future preclinical and clinical development of an AH-adjuvanted DCFHP vaccine candidate produced in CHO cells.

Decreased efficacy of a COVID-19 vaccine due to mutations present in early SARS-CoV-2 variants of concern.

With the SARS-CoV-2 virus still circulating and evolving, there remains an outstanding question if variant-specific vaccines represent the optimal path forward, or if other strategies might be more efficacious towards providing broad protection against emerging variants. Here, we examine the efficacy of strain-specific variants of our previously reported, pan-sarbecovirus vaccine candidate, DCFHP-alum, a ferritin nanoparticle functionalized with an engineered form of the SARS-CoV-2 spike protein. In non-human primates, DCFHP-alum elicits neutralizing antibodies against all known VOCs that have emerged to date and SARS-CoV-1. During development of the DCFHP antigen, we investigated the incorporation of strain-specific mutations from the major VOCs that had emerged to date: D614G, Epsilon, Alpha, Beta, and Gamma. Here, we report the biochemical and immunological characterizations that led us to choose the ancestral Wuhan-1 sequence as the basis for the final DCFHP antigen design. Specifically, we show by size exclusion chromatography and differential scanning fluorimetry that mutations in the VOCs adversely alter the antigen's structure and stability. More importantly, we determined that DCFHP without strain-specific mutations elicits the most robust, cross-reactive response in both pseudovirus and live virus neutralization assays. Our data suggest potential limitations to the variant-chasing approach in the development of protein nanoparticle vaccines, but also have implications for other approaches including mRNA-based vaccines.

Formulation development and comparability studies with an aluminum-salt adjuvanted SARS-CoV-2 Spike ferritin nanoparticle vaccine antigen produced from two different cell lines.

The development of safe and effective second-generation COVID-19 vaccines to improve affordability and storage stability requirements remains a high priority to expand global coverage. In this report, we describe formulation development and comparability studies with a self-assembled SARS-CoV-2 spike ferritin nanoparticle vaccine antigen (called DCFHP), when produced in two different cell lines and formulated with an aluminum-salt adjuvant (Alhydrogel, AH). Varying levels of phosphate buffer altered the extent and strength of antigen-adjuvant interactions, and these formulations were evaluated for their (1) in vivo performance in mice and (2) in vitro stability profiles. Unadjuvanted DCFHP produced minimal immune responses while AH-adjuvanted formulations elicited greatly enhanced pseudovirus neutralization titers independent of ∼100%, ∼40% or ∼10% of the DCFHP antigen adsorbed to AH. These formulations differed, however, in their in vitro stability properties as determined by biophysical studies and a competitive ELISA for measuring ACE2 receptor binding of AH-bound antigen. Interestingly, after one month of 4°C storage, small increases in antigenicity with concomitant decreases in the ability to desorb the antigen from the AH were observed. Finally, we performed a comparability assessment of DCFHP antigen produced in Expi293 and CHO cells, which displayed expected differences in their N-linked oligosaccharide profiles. Despite consisting of different DCFHP glycoforms, these two preparations were highly similar in their key quality attributes including molecular size, structural integrity, conformational stability, binding to ACE2 receptor and mouse immunogenicity profiles. Taken together, these studies support future preclinical and clinical development of an AH-adjuvanted DCFHP vaccine candidate produced in CHO cells.

A ferritin-based COVID-19 nanoparticle vaccine that elicits robust, durable, broad-spectrum neutralizing antisera in non-human primates.

While the rapid development of COVID-19 vaccines has been a scientific triumph, the need remains for a globally available vaccine that provides longer-lasting immunity against present and future SARS-CoV-2 variants of concern (VOCs). Here, we describe DCFHP, a ferritin-based, protein-nanoparticle vaccine candidate that, when formulated with aluminum hydroxide as the sole adjuvant (DCFHP-alum), elicits potent and durable neutralizing antisera in non-human primates against known VOCs, including Omicron BQ.1, as well as against SARS-CoV-1. Following a booster ~one year after the initial immunization, DCFHP-alum elicits a robust anamnestic response. To enable global accessibility, we generated a cell line that can enable production of thousands of vaccine doses per liter of cell culture and show that DCFHP-alum maintains potency for at least 14 days at temperatures exceeding standard room temperature. DCFHP-alum has potential as a once-yearly (or less frequent) booster vaccine, and as a primary vaccine for pediatric use including in infants.

HIV-1 prehairpin intermediate inhibitors show efficacy independent of neutralization tier.

HIV-1 strains are categorized into one of three neutralization tiers based on the relative ease by which they are neutralized by plasma from HIV-1-infected donors not on antiretroviral therapy; tier-1 strains are particularly sensitive to neutralization while tier-2 and tier-3 strains are increasingly difficult to neutralize. Most broadly neutralizing antibodies (bnAbs) previously described target the native prefusion conformation of HIV-1 Envelope (Env), but the relevance of the tiered categories for inhibitors targeting another Env conformation, the prehairpin intermediate, is not well understood. Here, we show that two inhibitors targeting distinct highly conserved regions of the prehairpin intermediate have strikingly consistent neutralization potencies (within ~100-fold for a given inhibitor) against strains in all three neutralization tiers of HIV-1; in contrast, best-in-class bnAbs targeting diverse Env epitopes vary by more than 10,000-fold in potency against these strains. Our results indicate that antisera-based HIV-1 neutralization tiers are not relevant for inhibitors targeting the prehairpin intermediate and highlight the potential for therapies and vaccine efforts targeting this conformation.

A ferritin-based COVID-19 nanoparticle vaccine that elicits robust, durable, broad-spectrum neutralizing antisera in non-human primates.

While the rapid development of COVID-19 vaccines has been a scientific triumph, the need remains for a globally available vaccine that provides longer-lasting immunity against present and future SARS-CoV-2 variants of concern (VOCs). Here, we describe DCFHP, a ferritin-based, protein-nanoparticle vaccine candidate that, when formulated with aluminum hydroxide as the sole adjuvant (DCFHP-alum), elicits potent and durable neutralizing antisera in non-human primates against known VOCs, including Omicron BQ.1, as well as against SARS-CoV-1. Following a booster ∻one year after the initial immunization, DCFHP-alum elicits a robust anamnestic response. To enable global accessibility, we generated a cell line that can enable production of thousands of vaccine doses per liter of cell culture and show that DCFHP-alum maintains potency for at least 14 days at temperatures exceeding standard room temperature. DCFHP-alum has potential as a once-yearly booster vaccine, and as a primary vaccine for pediatric use including in infants.

Adaption of a conventional ELISA to a 96-well ELISA-Array for measuring the antibody responses to influenza virus proteins and vaccines.

We describe an adaptation of conventional ELISA methods to an ELISA-Array format using non-contact Piezo printing of up to 30 spots of purified recombinant viral fusion proteins and vaccine on 96 well high-protein binding plates. Antigens were printed in 1 nanoliter volumes of protein stabilizing buffer using as little as 0.25 nanograms of protein, 2000-fold less than conventional ELISA. The performance of the ELISA-Array was demonstrated by serially diluting n = 9 human post-flu vaccination plasma samples starting at a 1/1000 dilution and measuring binding to the array of Influenza antigens. Plasma polyclonal antibody levels were detected using a cocktail of biotinylated anti-human kappa and lambda light chain antibodies, followed by a Streptavidin-horseradish peroxidase conjugate and the dose-dependent signal was developed with a precipitable TMB substrate. Intra- and inter-assay precision of absorbance units among the eight donor samples showed mean CVs of 4.8% and 10.8%, respectively. The plasma could be differentiated by donor and antigen with titer sensitivities ranging from 1 × 103 to 4 × 106, IC50 values from 1 × 104 to 9 × 106, and monoclonal antibody sensitivities in the ng/mL range. Equivalent sensitivities of ELISA versus ELISA-Array, compared using plasma and an H1N1 HA trimer, were achieved on the ELISA-Array printed at 0.25 ng per 200um spot and 1000 ng per ELISA 96-well. Vacuum-sealed array plates were shown to be stable when stored for at least 2 days at ambient temperature and up to 1 month at 4-8 °C. By the use of any set of printed antigens and analyte matrices the methods of this multiplexed ELISA-Array format can be broadly applied in translational research.

Light regulation of light-harvesting antenna size substantially enhances photosynthetic efficiency and biomass yield in green algae†.

One of the major factors limiting biomass productivity in algae is the low thermodynamic efficiency of photosynthesis. The greatest thermodynamic inefficiencies in photosynthesis occur during the conversion of light into chemical energy. At full sunlight the light-harvesting antenna captures photons at a rate nearly 10 times faster than the rate-limiting step in photosynthetic electron transport. Excess captured energy is dissipated by non-productive pathways including the production of reactive oxygen species. Substantial improvements in photosynthetic efficiency have been achieved by reducing the optical cross-section of the light-harvesting antenna by selectively reducing chlorophyll b levels and peripheral light-harvesting complex subunits. Smaller light-harvesting antenna, however, may not exhibit optimal photosynthetic performance in low or fluctuating light environments. We describe a translational control system to dynamically adjust light-harvesting antenna sizes for enhanced photosynthetic performance. By expressing a chlorophyllide a oxygenase (CAO) gene having a 5' mRNA extension encoding a Nab1 translational repressor binding site in a CAO knockout line it was possible to continuously alter chlorophyll b levels and correspondingly light-harvesting antenna sizes by light-activated Nab1 repression of CAO expression as a function of growth light intensity. Significantly, algae having light-regulated antenna sizes had substantially higher photosynthetic rates and two-fold greater biomass productivity than the parental wild-type strains as well as near wild-type ability to carry out state transitions and non-photochemical quenching. These results have broad implications for enhanced algae and plant biomass productivity.

Functional Enrichment and Analysis of Antigen-Specific Memory B Cell Antibody Repertoires in PBMCs.

Phenotypic screening of antigen-specific antibodies in human blood is a common diagnostic test for infectious agents and a correlate of protection after vaccination. In addition to long-lived antibody secreting plasma cells residing in the bone marrow, memory B cells are a latent source of antigen-experienced, long-term immunity that can be found at low frequencies in circulating peripheral blood mononuclear cells (PBMCs). Assessing the genotype, clonal frequency, quality, and function of antibodies resulting from an individual's persistent memory B cell repertoire can help inform the success or failure of immune protection. Using in vitro polyclonal stimulation, we functionally expand the memory repertoire from PBMCs and clonally map monoclonal antibodies from this population. We show that combining deep sequencing of stimulated memory B cell repertoires with retrieving single antigen-specific cells is a promising approach in evaluating the latent, functional B cell memory in PBMCs.

Domain orientation in the inactive response regulator Mycobacterium tuberculosis MtrA provides a barrier to activation.

The structure of MtrA, an essential gene product for the human pathogen Mycobacterium tuberculosis, has been solved to a resolution of 2.1 A. MtrA is a member of the OmpR/PhoB family of response regulators and represents the fourth family member for which a structure of the protein in its inactive state has been determined. As is true for all OmpR/PhoB family members, MtrA possesses an N-terminal regulatory domain and a C-terminal winged helix-turn-helix DNA-binding domain, with phosphorylation of the regulatory domain modulating the activity of the protein. In the inactive form of MtrA, these two domains form an extensive interface that is composed of the alpha4-beta5-alpha5 face of the regulatory domain and the C-terminal end of the positioning helix, the trans-activation loop, and the recognition helix of the DNA-binding domain. This domain orientation suggests a mechanism of mutual inhibition by the two domains. Activation of MtrA would require a disruption of this interface to allow the alpha4-beta5-alpha5 face of the regulatory domain to form the intermolecule interactions that are associated with the active state and to allow the recognition helix to interact with DNA. Furthermore, the interface appears to stabilize the inactive conformation of MtrA, potentially reducing the rate of phosphorylation of the N-terminal domain. This combination of effects may form a switch, regulating the activity of MtrA. The domain orientation exhibited by MtrA also provides a rationale for the variation in linker length that is observed within the OmpR/PhoB family of response regulators.

Structure of a cholesterol-binding protein deficient in Niemann-Pick type C2 disease.

Niemann-Pick disease type C2 (NP-C2) is a fatal hereditary disease characterized by accumulation of low-density lipoprotein-derived cholesterol in lysosomes. Here we report the 1.7-A resolution crystal structure of the cholesterol-binding protein deficient in this disease, NPC2, and the characterization of its ligand binding properties. Human NPC2 binds the cholesterol analog dehydroergosterol with submicromolar affinity at both acidic and neutral pH. NPC2 has an Ig-like fold stabilized by three disulfide bonds. The structure of the bovine protein reveals a loosely packed region penetrating from the surface into the hydrophobic core that forms adjacent small cavities with a total volume of approximately 160 A(3). We propose that this region represents the incipient cholesterol-binding site that dilates to accommodate an approximately 740-A(3) cholesterol molecule.