Understanding structure activity relationships of Good HEPES lipids for lipid nanoparticle mRNA vaccine applications.

Lipid nanoparticles (LNPs) have shown great promise as delivery vehicles to transport messenger ribonucleic acid (mRNA) into cells and act as vaccines for infectious diseases including COVID-19 and influenza. The ionizable lipid incorporated within the LNP is known to be one of the main driving factors for potency and tolerability. Herein, we describe a novel family of ionizable lipids synthesized with a piperazine core derived from the HEPES Good buffer. These ionizable lipids have unique asymmetric tails and two dissimilar degradable moieties incorporated within the structure. Lipids tails of varying lengths, degrees of unsaturation, branching, and the inclusion of additional ester moieties were evaluated for protein expression. We observed several key lipid structure activity relationships that correlated with improved protein production in vivo, including lipid tails of 12 carbons on the ester side and the effect of carbon spacing on the disulfide arm of the lipids. Differences in LNP physical characteristics were observed for lipids containing an extra ester moiety. The LNP structure and lipid bilayer packing, visualized through Cryo-TEM, affected the amount of protein produced in vivo. In non-human primates, the Good HEPES LNPs formulated with an mRNA encoding an influenza hemagglutinin (HA) antigen successfully generated functional HA inhibition (HAI) antibody titers comparable to the industry standards MC3 and SM-102 LNPs, demonstrating their promise as a potential vaccine.

Effects of esterification, saturation and amount of fatty acids infused into the rumen or abomasum in lactating dairy cows.

Our objective was to determine the effects of chemical structure, amount, and site of infusion of long-chain fatty acids (LCFA) in lactating dairy cows. Six multiparous Holstein cows were used in a 6 × 6 Latin square design with 21-d periods. During d 1 to 14, 250 g/d of LCFA and during d 15 to 21, 500 g/d of LCFA were infused continuously into either the rumen or abomasum. Treatments were 1) Control (CONT); 200 g/d of meat solubles plus 12 g/d of Tween 80 in 10 L of water, administered half in the rumen and half in abomasum; 2) control plus mostly saturated LCFA into the abomasum (SFAA); 3) control plus mostly saturated LCFA into the rumen (SFAR); 4) control plus soy (mostly unsaturated LCFA) free fatty acids (FFA) into the abomasum (UFAA); 5) control plus soy triglycerides (TG) into the abomasum (TGA); and 6) control plus soy TG into the rumen (TGR). The first 10 d of each period were for adaptation and washout from the previous treatment. The diet consisted of 30% (dry matter basis) corn silage, 20% alfalfa silage and 50% concentrate. Cows infused with UFAA had lower dry matter intake and milk yield than those infused with SFAA or TGA and reductions were greater at the higher infusion amount. Milk fat yield was decreased by UFAA relative to other treatments. Unsaturated LCFA decreased milk fat yield more than saturated LCFA. All LCFA treatments decreased short- and medium-chain FA in milk relative to CONT, with greatest decreases for UFAA. Apparent total tract digestibilities of nutrient fractions were decreased by UFAA compared with TGA and SFAA and tended to be lower at the higher infusion amount. Apparent digestibility of total fatty acids (FA) was greater for SFAR than for SFAA. Plasma glucagon-like peptide-1 was greater for cows infused with UFAA than SFAA or TGA and increased at the higher amount. Plasma cholecystokinin was greater for cows infused with LCFA compared with CONT. Postruminal unsaturated FFA reduced intake and digestibility of nutrients and FA compared with postruminal TG infusion; saturated FA did not decrease dry matter intake or disrupt nutrient digestion. Glucagon-like peptide-1 may be involved in regulation of feed intake by long-chain fatty acids.

Pan-SARS neutralizing responses after third boost vaccination in non-human primate immunogenicity model.

The emergence of SARS-CoV-2 variants, especially Beta and Delta, has raised concerns about the reduced protection from previous infection or vaccination based on the original Wuhan-Hu-1 (D614) virus. To identify promising regimens for inducing neutralizing titers towards new variants, we evaluated monovalent and bivalent mRNA vaccines either as primary vaccination or as a booster in nonhuman primates (NHPs). Two mRNA vaccines, D614-based MRT5500 and Beta-based MRT5500ß, tested in sequential regimens or as a bivalent combination in naïve NHPs produced modest neutralizing titers to heterologous variants. However, when mRNA vaccines were administered as a booster to pre-immune NHPs, we observed a robust increase in neutralizing titers with expanded breadth towards all tested variants, and notably SARS-CoV-1. The breadth of the neutralizing response was independent of vaccine sequence or modality, as we further showed either MRT5500 or recombinant subunit Spike protein (with adjuvant) can serve as boosters to induce broadly neutralizing antibodies in the NHPs primed with MRT5500. The data support the notion that a third vaccination is key to boosting existing titers and improving the breadth of antibodies to address variants of concern, including those with an E484K mutation in the Receptor Binding Domain (RBD) (Beta, Gamma).

Immunogenicity and efficacy of mRNA COVID-19 vaccine MRT5500 in preclinical animal models.

Emergency use authorization of COVID vaccines has brought hope to mitigate pandemic of coronavirus disease 2019 (COVID-19). However, there remains a need for additional effective vaccines to meet the global demand and address the potential new viral variants. mRNA technologies offer an expeditious path alternative to traditional vaccine approaches. Here we describe the efforts to utilize an mRNA platform for rational design and evaluations of mRNA vaccine candidates based on the spike (S) glycoprotein of SARS-CoV-2. Several mRNA constructs of S-protein, including wild type, a pre-fusion stabilized mutant (2P), a furin cleavage-site mutant (GSAS) and a double mutant form (2P/GSAS), as well as others, were tested in animal models for their capacity to elicit neutralizing antibodies (nAbs). The lead 2P/GSAS candidate was further assessed in dose-ranging studies in mice and Cynomolgus macaques, and for efficacy in a Syrian golden hamster model. The selected 2P/GSAS vaccine formulation, designated MRT5500, elicited potent nAbs as measured in neutralization assays in all three preclinical models and more importantly, protected against SARS-CoV-2-induced weight loss and lung pathology in hamsters. In addition, MRT5500 elicited TH1-biased responses in both mouse and non-human primate (NHP), thus alleviating a hypothetical concern of potential vaccine-associated enhanced respiratory diseases known associated with TH2-biased responses. These data position MRT5500 as a viable vaccine candidate for entering clinical development.

Pork as a Source of Omega-3 (n-3) Fatty Acids.

Pork is the most widely eaten meat in the world, but typical feeding practices give it a high omega-6 (n-6) to omega-3 (n-3) fatty acid ratio and make it a poor source of n-3 fatty acids. Feeding pigs n-3 fatty acids can increase their contents in pork, and in countries where label claims are permitted, claims can be met with limited feeding of n-3 fatty acid enrich feedstuffs, provided contributions of both fat and muscle are included in pork servings. Pork enriched with n-3 fatty acids is, however, not widely available. Producing and marketing n-3 fatty acid enriched pork requires regulatory approval, development costs, quality control costs, may increase production costs, and enriched pork has to be tracked to retail and sold for a premium. Mandatory labelling of the n-6/n-3 ratio and the n-3 fatty acid content of pork may help drive production of n-3 fatty acid enriched pork, and open the door to population-based disease prevention polices (i.e., food tax to provide incentives to improve production practices). A shift from the status-quo, however, will require stronger signals along the value chain indicating production of n-3 fatty acid enriched pork is an industry priority.