Microscopic Rates of Peptide–Phospholipid Bilayer Interactions from Single-Molecule Residence Times

The binding of glucagon-like peptide-1 (GLP-1) to a planar phospholipid bilayer is measured using single-molecule total internal reflection fluorescence microscopy. From several reports in the literature, GLP-1 has been shown to be a random coil in free solution, adopting a folded, α-helix conformation when intercalated into membrane environments. Single-molecule fluorescence measurements of GLP-1 binding to supported lipid bilayers show evidence of two populations of membrane-associated molecules having different residence times, suggesting weakly adsorbed peptides and strongly bound peptides in the lipid bilayer. The path to and from a strongly bound (folded, intercalated) state would likely include an adsorbed state as an intermediate, so that the resulting kinetics would correspond to a consecutive first-order reversible three-state model. In this work, the relationships between measured single-molecule residence times and the microscopic rates in a three-state kinetic model are derived and used to interpret the binding of GLP-1 to a supported lipid bilayer. The system of differential equations associated with the proposed consecutive-three state kinetics scheme is solved, and the solution is applied to interpret histograms of single-molecule, GLP-1 residence times in terms of the microscopic rates in the sequential two-step model. These microscopic rates are used to estimate the free energy barrier to adsorption, the fraction of peptides adsorbing to the membrane surface that successfully intercalate in the bilayer, the lifetime of inserted peptides in the membrane, and the free energy change of insertion into the lipid bilayer from the adsorbed state. The transition from a random coil in solution to a folded state in a membrane has been recognized as a common motif for insertion of membrane active peptides. Therefore, the relationships developed here could have wide application to the kinetic analysis of peptide–membrane interactions

GLA and CpG combine to enhance the protective efficacy of ID93.

<p>Mice were immunized and challenged with a low dose of aerosolized <i>M.tb.</i> (A) four or (B) twelve weeks later. <i>M.tb.</i> burdens in the lungs were determined four weeks after infection. Statistically significant differences between vaccinated groups were determined by ANOVA using the Bonferroni correction for multiple comparisons. Data are representative of three experiments with similar results with seven animals per group.</p

Enhanced T_H1 responses to GLA and CpG are independent of TRIF signaling.

<p>(A) C57BL/6 and TRIF^−/− splenocytes were stimulated <i>ex vivo</i> with GLA, CpG or both and analyzed for IL-6, IL-12p40, and TNF production by macrophages. (B and C) C57BL/6 and TRIF^−/− mice were immunized with ID93 adjuvanted with GLA, CpG or GLA+CpG. Splenic ID93-specific T_H1 CD⁺4 T cells from immunized mice were identified by cytokine production following ex-vivo restimulation with ID93. Data are representative of two experiments with similar results with 3–5 animals per group. ^*,^**,^***, and ^**** indicate <i>P</i><0.05, 0.01, 0.001, and 0.0001 respectively, relative to GLA+CpG as determined by ANOVA using the Bonferroni correction for multiple comparisons.</p

GLA and CpG combine to enhance T_H1 responses to ID93.

<p>Mice were immunized with ID93 adjuvanted with GLA, CpG, or GLA+CpG. (A and B) One week after the final immunization ID93-specific CD4⁺ T cells were identified by staining with I-A^b tetramers presenting dominant epitopes from Rv2608 and Rv3619. Cells are gated as singlet, CD4⁺ CD44⁺. Splenic ID93-specific T_H1 CD4⁺ T cells from immunized mice were identified by cytokine production following <i>ex-vivo</i> restimulation with ID93 and analyzed for (C) total cytokine response or (D) poly-functional responses. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083884#pone.0083884.s001" target="_blank">Figure S1</a> for representative cytokine staining. Data are representative of four experiments with similar results with 3–5 animals per group. ^*,^**,^***, and ^**** indicate <i>P</i><0.05, 0.01, 0.001, and 0.0001 respectively, relative to GLA+CpG as determined by ANOVA using the Bonferroni correction for multiple comparisons.</p

DataSheet1_Choice of adjuvant and antigen composition alters the immunogenic profile of a SARS-CoV-2 subunit vaccine.PDF

Introduction: Since their introduction, adjuvanted recombinant subunit vaccines against COVID-19 have played a pivotal role in protecting global populations. Optimizing the immune response’s quality, amplitude, and durability to these vaccines depends on the appropriate adjuvant choice and dose in combination with the selected antigen.Methods: Here, we employed a preclinical mouse model to study the adaptive humoral and cellular immune responses to a SARS-CoV-2 receptor binding domain (RBD) antigen formulated with one of four different immune agonists [GLA, 3M-052, CpG-1826 (CpG), and dmLT], in combination with one of two different immune-stimulating formulations, a stabilized squalene emulsion (SE) or aluminum hydroxide (Alum). Using a weighted desirability index, we established an immunogenicity ranking for each adjuvant in combination with the RBD antigen.Results: We found that formulations of the RBD with Alum in combination with either 3M-052 or CpG led to at least a 2-log increase in serum IgG production and a 1.3- to 2.2-log increase in the number of bone marrow-derived antibody-secreting cells compared to the RBD formulated with Alum without an additional agonist. In contrast, the RBD formulated with SE in combination with 3M-052 or CpG did not elicit an IgG response greater than the unadjuvanted control. Additionally, RBD formulated with 3M-052 or CpG on Alum generated a 0.8- or 1.6-log lower splenocyte IL-5 response (a pro-Th2 marker), respectively, than Alum without an additional agonist. When formulated with 3M-052-Alum, a bivalent vaccine containing the original lineage (Wuhan-Hu-1) and the Delta variant (B.1.617.2) RBD antigens led to a more than 2-log increase in neutralizing antibodies against an Omicron variant (B.1.1.529) pseudovirus in vaccinated animals compared to animals that received the monovalent RBD antigen.Discussion: Our results suggest that optimal immune responses to subunit antigens may be achieved through an orthogonal approach that applies adjuvant formulation, antigen combination, and advances in rational vaccine development techniques.</p

rCSP/Ad35CS Heterologous Prime-Boost Regimens.

<p>C57BL/6 mice were immunized with rCSP formulated in GLA-SE or Ad35CS according to the regimens shown.</p><p>rCSP/Ad35CS Heterologous Prime-Boost Regimens.</p

DataSheet1_Lyophilization process engineering and thermostability of ID93 + GLA-SE, a single-vial adjuvanted subunit tuberculosis vaccine candidate for use in clinical studies.pdf

Promising clinical efficacy results have generated considerable enthusiasm for the potential impact of adjuvant-containing subunit tuberculosis vaccines. The development of a thermostable tuberculosis vaccine formulation could have significant benefits on both the cost and feasibility of global vaccine distribution. The tuberculosis vaccine candidate ID93 + GLA-SE has reached Phase 2 clinical testing, demonstrating safety and immunogenicity as a two-vial point-of-care mixture. Earlier publications have detailed efforts to develop a lead candidate single-vial lyophilized thermostable ID93 + GLA-SE vaccine formulation. The present report describes the lyophilization process development and scale-up of the lead candidate thermostable ID93 + GLA-SE composition. The manufacture of three full-scale engineering batches was followed by one batch made and released under current Good Manufacturing Practices (cGMP). Up to 4.5 years of stability data were collected. The cGMP lyophilized ID93 + GLA-SE passed all manufacturing release test criteria and maintained stability for at least 3 months when stored at 37°C and up to 24 months when stored at 5°C. This work represents the first advancement of a thermostable adjuvant-containing subunit tuberculosis vaccine to clinical testing readiness.</p

Immunogenicity and Efficacy of rCSP Immunization in Mice.

<p>Five C57BL/6 mice per cohort were immunized 3x with 25 µg of rCSP from 3 production strains (CSP-1, CSP-2, and CSP-3) formulated in incomplete Freund's adjuvant. Sera were collected 2 weeks after the last immunization and pooled for analysis by ELISA, IFA, and ISI. Mice were challenged with <i>Pb</i>-CS(<i>Pf</i>) parasites 3 weeks after the last immunization. (<b>A</b>) Pooled sera from mice immunized with the 3 recombinant proteins showed similar ELISA and IFA titers. ELISA titers to a [NANP]₆ peptide were calculated at OD = 1.0 based on 4-parameter logistic curve fits, and IFA titers were determined based on the lowest serum dilution to give sporozoites-specific fluorescence above background level shown by sera from naïve mice. (<b>B</b>) Sera from rCSP-immunized mice reacted to <i>Pb</i>-CS(<i>Pf</i>) sporozoites demonstrated the expected pattern of surface fluorescence by IFA. (<b>C</b>) Sera from rCSP-immunized mice diluted 1∶100 and assayed for the ability to block <i>Pf</i> sporozoite infection of hepatocytes in an ISI assay demonstrated the highest ISI activity for anti-CSP-1 protein sera. The percent inhibition is shown relative to the number of sporozoites after incubating with the sera of naïve mice. Percent inhibition of LS parasite development in rCSP-immunized mice challenged with <i>Pb</i>-CS(<i>Pf</i>) sporozoites normalized to naïve control mice is also shown. The reduction of LS parasites in livers of mice immunized with CSP-1 or CSP-3 proteins was statistically significant based on the mean parasite-specific 18 s rRNA copy number compared the level of LS parasites in livers of naïve and adjuvant-only administered mice (as noted by asterisks).</p

rCSP Strain Selection and Identification.

<p>(<b>A</b>) Reduced SDS-PAGE analysis of lysate samples prior to purification. Lanes 1–3 represent samples from strain CSP-1, CSP-3, and CSP-2 fermentations, respectively. Lane 4 represents a sample from a culture of <i>P. fluorescens</i> transformed with plasmid vector containing no insert. Arrow denotes rCSP in lysate samples. (<b>B</b>) Reduced SDS-PAGE analysis of purified proteins from a 2-column purification process. Lanes 1–3 represent samples from CSP-1, CSP-2, and CSP-3 productions, respectively. (<b>C</b>) Western blot of non-reduced purified CSP-1, CSP-2, and CSP-3 proteins (lanes 2–4, respectively). Lane 1 represents a sample from a culture of <i>P. fluorescens</i> transformed with plasmid vector containing no insert.</p

Sterile Protection with rCSP.

<p>BALB/c mice were immunized 3x with 25 µg of rCSP formulated in GLA-SE or GLA-LSQ, followed by challenge with Pb-CS(Pf) parasites 2 weeks after the last immunization. Parasitemia was monitored by Giemsa-stained blood smears daily from 7 to 11 days post challenge. Animals with no detectable parasitemia on all days monitored were considered sterilely protected.</p><p>Sterile Protection with rCSP.</p