A Cysteine Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus

Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity. We recently developed a vaccine antigen against respiratory syncytial virus (RSV), which comprised the fusion (F) glycoprotein stabilized in its pre-fusion trimeric conformation by “DS-Cav1” mutations and by an appended C-terminal trimerization motif or “foldon” from T4-bacteriophage fibritin. Here we investigate the creation of a cyste- ine zipper to allow for the removal of the phage foldon, while maintaining the immunogenic- ity of the parent DS-Cav1+foldon antigen. Constructs without foldon yielded RSV F monomers, and enzymatic removal of the phage foldon from pre-fusion F trimers resulted in their dissociation into monomers. Because the native C terminus of the pre-fusion RSV F ectodomain encompasses a viral trimeric coiled-coil, we explored whether introduction of cysteine residues capable of forming inter-protomer disulfides might allow for stable trimers. Structural modeling indicated the introduced cysteines to form disulfide “rings”, with each ring comprising a different set of inward facing residues of the coiled-coil. Three sets of rings could be placed within the native RSV F coiled-coil, and additional rings could be added by duplicating portions of the coiled-coil. High levels of neutralizing activity in mice, equivalent to that of the parent DS-Cav1+foldon antigen, were elicited by a 4-ring stabilized RSV F trimer with no foldon. Structure-based alteration of a viral coiled-coil to create a cys- teine zipper thus allows a phage trimerization motif to be removed from a candidate vaccine antigen

Mapping monoclonal anti-SARS-CoV-2 antibody repertoires against diverse coronavirus antigens

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged continuously, challenging the effectiveness of vaccines, diagnostics, and treatments. Moreover, the possibility of the appearance of a new betacoronavirus with high transmissibility and high fatality is reason for concern. In this study, we used a natively paired yeast display technology, combined with next-generation sequencing (NGS) and massive bioinformatic analysis to perform a comprehensive study of subdomain specificity of natural human antibodies from two convalescent donors. Using this screening technology, we mapped the cross-reactive responses of antibodies generated by the two donors against SARS-CoV-2 variants and other betacoronaviruses. We tested the neutralization potency of a set of the cross-reactive antibodies generated in this study and observed that most of the antibodies produced by these patients were non-neutralizing. We performed a comparison of the specific and non-specific antibodies by somatic hypermutation in a repertoire-scale for the two individuals and observed that the degree of somatic hypermutation was unique for each patient. The data from this study provide functional insights into cross-reactive antibodies that can assist in the development of strategies against emerging SARS-CoV-2 variants and divergent betacoronaviruses

Adjuvants and the vaccine response to the DS-Cav1-stabilized fusion glycoprotein of respiratory syncytial virus

Appropriate adjuvant selection may be essential to optimize the potency and to tailor the immune response of subunit vaccines. To induce protective responses against respiratory syncytial virus (RSV)—a highly prevalent childhood pathogen without a licensed vaccine—we previously engineered a pre-fusion-stabilized trimeric RSV F (pre-F) “DS-Cav1” immunogen, which induced high titer RSV-neutralizing antibodies, in mice and non-human primates, when formulated with adjuvants Poly (I:C) and Poly (IC:LC), respectively. To assess the impact of different adjuvants, here we formulated RSV F DS-Cav1 with multiple adjuvants and assessed immune responses. Very high RSV-neutralizing antibody responses (19,006 EC50) were observed in naïve mice immunized with 2 doses of DS-Cav1 adjuvanted with Sigma adjuvant system (SAS), an oil-in-water adjuvant, plus Carbopol; high responses (3658–7108) were observed with DS-Cav1 adjuvanted with Alum, SAS alone, Adjuplex, Poly (I:C) and Poly (IC:LC); and moderate responses (1251–2129) were observed with DS-Cav1 adjuvanted with the TLR4 agonist MPLA, Alum plus MPLA or AddaVax. In contrast, DS-Cav1 without adjuvant induced low-level responses (6). A balanced IgG1 and IgG2a (Th2/Th1) immune response was elicited in most of the high to very high response groups (all but Alum and Adjuplex). We also tested the immune response induced by DS-Cav1 in elderly mice with pre-existing DS-Cav1 immunity; we observed that DS-Cav1 adjuvanted with SAS plus Carbopol boosted the response 2-3-fold, whereas DS-Cav1 adjuvanted with alum boosted the response 5-fold. Finally, we tested whether a mixture of ISA 71 VG and Carbopol would enhanced the antibody response in DS-Cav1 immunized calves. While pre-F-stabilized bovine RSV F induced very high titers in mice when adjuvanted with SAS plus Carbopol, the addition of Carbopol to ISA 71 VG did not enhance immune responses in calves. The vaccine response to pre-F-stabilized RSV F is augmented by adjuvant, but the degree of adjuvant-induced enhancement appears to be both context-dependent and species-specific

Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy

NMR spectroscopic characterization of the structure or the dynamics of proteins generally requires the production of samples isotopically enriched in 15N, 13C, or 2H. The bacterial expression systems currently in use to obtain isotopic enrichment, however, cannot produce a number of eukaryotic proteins, especially those that require post-translational modifications such as N-linked glycosylation for proper folding or activity. Here, we report the use of an adenovirus vector-based mammalian expression system to produce isotopically enriched 15N or 15N/13C samples of an outer domain variant of the HIV-1 gp120 envelope glycoprotein with 15 sites of N-linked glycosylation. Yields for the 15N- and 15N/13C-labeled gp120s after affinity chromatography were 45 and 44 mg/l, respectively, with an average of over 80% isotope incorporation. Recognition of the labeled gp120 by cognate antibodies that recognize complex epitopes showed affinities comparable to the unlabeled protein. NMR spectra, including 1H-15N and 1H-13C HSQCs, 15N-edited NOESY-HSQC, and 3D HNCO, were of high quality, with signal-to-noise consistent with an efficient level of isotope incorporation, and with chemical shift dispersion indicative of a well-folded protein. The exceptional protein yields, good isotope incorporation, and ability to obtain well-folded post-translationally modified proteins make this mammalian system attractive for the production of isotopically enriched eukaryotic proteins for NMR spectroscopy

Image Analysis to Detect Phase Transition Temperatures of p-n Alkyl benzoic acids

Abstract: Using statistical image analysis, phase transition temperatures of thermo-tropic liquid crystals p -n Alkyl benzoic acids (nBA) where n = 5 to 10 are investigated. Textures of liquid crystals as a function of temperature are recorded using Polarizing Optical Microscope (POM) with hot stage and camera attachment. In this analysis, statistical parameters -mean, variance, skewness and kurtosis are used to compute the liquid crystal textures using MATLAB software. Temperature dependence of statistical parameters for the abrupt changes and fluctuations are related to the phase transitions of the sample. The results are compared with literature values

Transition Temperatures of Thermotropic Liquid Crystals from the Local Binary Gray Level Cooccurrence Matrix

This paper presents a method which combines the statistical analysis with texture structural analysis called Local Binary Gray Level Cooccurrence Matrix (LBGLCM) to investigate the phase transition temperatures of thermotropic p,n-alkyloxy benzoic acid (nOBA, n=4,6,8,10 and 12) liquid crystals. Textures of the homeotropically aligned liquid crystal compounds are recorded as a function of temperature using polarizing optical microscope attached to the hot stage and high resolution camera. In this method, second-order statistical parameters (contrast, energy, homogeneity, and correlation) are extracted from the LBGLCM of the textures. The changes associatedwiththe values of extracted parameters as a function of temperature are a helpful process to identify the phases and phase transition temperatures of the samples. Results obtained from this method have validity and are in good agreement with the literature

Adjuvants and the vaccine response to the DS-Cav1-stabilized fusion glycoprotein of respiratory syncytial virus.

Appropriate adjuvant selection may be essential to optimize the potency and to tailor the immune response of subunit vaccines. To induce protective responses against respiratory syncytial virus (RSV)-a highly prevalent childhood pathogen without a licensed vaccine-we previously engineered a pre-fusion-stabilized trimeric RSV F (pre-F) "DS-Cav1" immunogen, which induced high titer RSV-neutralizing antibodies, in mice and non-human primates, when formulated with adjuvants Poly (I:C) and Poly (IC:LC), respectively. To assess the impact of different adjuvants, here we formulated RSV F DS-Cav1 with multiple adjuvants and assessed immune responses. Very high RSV-neutralizing antibody responses (19,006 EC50) were observed in naïve mice immunized with 2 doses of DS-Cav1 adjuvanted with Sigma adjuvant system (SAS), an oil-in-water adjuvant, plus Carbopol; high responses (3658-7108) were observed with DS-Cav1 adjuvanted with Alum, SAS alone, Adjuplex, Poly (I:C) and Poly (IC:LC); and moderate responses (1251-2129) were observed with DS-Cav1 adjuvanted with the TLR4 agonist MPLA, Alum plus MPLA or AddaVax. In contrast, DS-Cav1 without adjuvant induced low-level responses (6). A balanced IgG1 and IgG2a (Th2/Th1) immune response was elicited in most of the high to very high response groups (all but Alum and Adjuplex). We also tested the immune response induced by DS-Cav1 in elderly mice with pre-existing DS-Cav1 immunity; we observed that DS-Cav1 adjuvanted with SAS plus Carbopol boosted the response 2-3-fold, whereas DS-Cav1 adjuvanted with alum boosted the response 5-fold. Finally, we tested whether a mixture of ISA 71 VG and Carbopol would enhanced the antibody response in DS-Cav1 immunized calves. While pre-F-stabilized bovine RSV F induced very high titers in mice when adjuvanted with SAS plus Carbopol, the addition of Carbopol to ISA 71 VG did not enhance immune responses in calves. The vaccine response to pre-F-stabilized RSV F is augmented by adjuvant, but the degree of adjuvant-induced enhancement appears to be both context-dependent and species-specific