An IQ Consortium Perspective on Best Practices for Bioanalytical and Immunogenicity Assessment Aspects of CAR-T and TCR-T Cellular Therapies Development.

CAR-T therapies have shown remarkable efficacy against hematological malignancies in the clinic over the last decade and new studies indicate that progress is being made to use these novel therapies to target solid tumors as well as treat autoimmune disease. Innovation in the field, including TCR-T, allogeneic or "off the shelf" CAR-T, and autoantigen/armored CAR-Ts are likely to increase the efficacy and applications of these therapies. The unique aspects of these cell-based therapeutics; patient-derived cells, intracellular expression, in vivo expansion, and phenotypic changes provide unique bioanalytical challenges to develop pharmacokinetic and immunogenicity assessments. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) Translational and ADME Sciences Leadership Group (TALG) has brought together a group of industry experts to discuss and consider these challenges. In this white paper, we present the IQ consortium perspective on the best practices and considerations for bioanalytical and immunogenicity aspects toward the optimal development of CAR-T and TCR-T cell therapies.

Association of E484K Spike Protein Mutation With Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection in Vaccinated Persons: Maryland, January-May 2021.

Among 9048 people infected with SARS-CoV-2 between January and May 2021 in Maryland, in regression-adjusted analysis, SARS-CoV-2 viruses carrying the spike protein mutation E484K were disproportionately prevalent among persons infected after full vaccination against COVID-19 compared with infected persons who were not fully vaccinated (aOR, 1.96; 95% CI: 1.36-2.83).

Complete Genome Sequences of 20 Nontyphoidal Salmonella Isolates from Rwanda.

Nontyphoidal Salmonella enterica strains are major foodborne pathogens with global public health importance. Foodborne salmonellosis can be life-threatening, especially in sub-Saharan Africa. We report the complete genome sequences of 20 nontyphoidal Salmonella enterica strains isolated in Rwanda. The reported 20 bacterial chromosomes and 8 plasmids each belong to 1 of 9 nontyphoidal Salmonella serotypes.

Structure of the Cellulose Synthase Complex of Gluconacetobacter hansenii at 23.4 Å Resolution.

Bacterial crystalline cellulose is used in biomedical and industrial applications, but the molecular mechanisms of synthesis are unclear. Unlike most bacteria, which make non-crystalline cellulose, Gluconacetobacter hansenii extrudes profuse amounts of crystalline cellulose. Its cellulose synthase (AcsA) exists as a complex with accessory protein AcsB, forming a 'terminal complex' (TC) that has been visualized by freeze-fracture TEM at the base of ribbons of crystalline cellulose. The catalytic AcsAB complex is embedded in the cytoplasmic membrane. The C-terminal portion of AcsC is predicted to form a translocation channel in the outer membrane, with the rest of AcsC possibly interacting with AcsD in the periplasm. It is thus believed that synthesis from an organized array of TCs coordinated with extrusion by AcsC and AcsD enable this bacterium to make crystalline cellulose. The only structural data that exist for this system are the above mentioned freeze-fracture TEM images, fluorescence microscopy images revealing that TCs align in a row, a crystal structure of AcsD bound to cellopentaose, and a crystal structure of PilZ domain of AcsA. Here we advance our understanding of the structural basis for crystalline cellulose production by bacterial cellulose synthase by determining a negative stain structure resolved to 23.4 Å for highly purified AcsAB complex that catalyzed incorporation of UDP-glucose into ß-1,4-glucan chains, and responded to the presence of allosteric activator cyclic diguanylate. Although the AcsAB complex was functional in vitro, the synthesized cellulose was not visible in TEM. The negative stain structure revealed that AcsAB is very similar to that of the BcsAB synthase of Rhodobacter sphaeroides, a non-crystalline cellulose producing bacterium. The results indicate that the crystalline cellulose producing and non-crystalline cellulose producing bacteria share conserved catalytic and membrane translocation components, and support the hypothesis that it is the extrusion mechanism and order in linearly arrayed TCs that enables production of crystalline cellulose.

In vitro synthesis of cellulose microfibrils by a membrane protein from protoplasts of the non-vascular plant Physcomitrella patens.

Plant cellulose synthases (CesAs) form a family of membrane proteins that are associated with hexagonal structures in the plasma membrane called CesA complexes (CSCs). It has been difficult to purify plant CesA proteins for biochemical and structural studies. We describe CesA activity in a membrane protein preparation isolated from protoplasts of Physcomitrella patens overexpressing haemagglutinin (HA)-tagged PpCesA5. Incubating the membrane preparation with UDP-glucose predominantly produced cellulose. Negative-stain EM revealed microfibrils. Cellulase bound to and degraded these microfibrils. Vibrational sum frequency generation (SFG) spectroscopic analysis detected the presence of crystalline cellulose in the microfibrils. Putative CesA proteins were frequently observed attached to the microfibril ends. Combined cross-linking and gradient centrifugation showed bundles of cellulose microfibrils with larger particle aggregates, possibly CSCs. These results suggest that P. patens is a useful model system for biochemical and structural characterization of plant CSCs and their components.

MreA functions in the global regulation of methanogenic pathways in Methanosarcina acetivorans.

UNLABELLED: Results are presented supporting a regulatory role for the product of the MA3302 gene locus (designated MreA) previously annotated as a hypothetical protein in the methanogenic species Methanosarcina acetivorans of the domain Archaea. Sequence analysis of MreA revealed identity to the TrmB family of transcription factors, albeit the sequence is lacking the sensor domain analogous to TrmBL2, abundant in nonmethanogenic species of the domain Archaea. Transcription of mreA was highly upregulated during growth on acetate versus methylotrophic substrates, and an mreA deletion (ΔmreA) strain was impaired for growth with acetate in contrast to normal growth with methylotrophic substrates. Transcriptional profiling of acetate-grown cells identified 280 genes with altered expression in the ΔmreA strain versus the wild-type strain. Expression of genes unique to the acetate pathway decreased whereas expression of genes unique to methylotrophic metabolism increased in the ΔmreA strain relative to the wild type, results indicative of a dual role for MreA in either the direct or indirect activation of acetate-specific genes and repression of methylotrophic-specific genes. Gel shift experiments revealed specific binding of MreA to promoter regions of regulated genes. Homologs of MreA were identified in M. acetivorans and other Methanosarcina species for which expression patterns indicate roles in regulating methylotrophic pathways. IMPORTANCE: Species in the domain Archaea utilize basal transcription machinery resembling that of the domain Eukarya, raising questions addressing the role of numerous putative transcription factors identified in sequenced archaeal genomes. Species in the genus Methanosarcina are ideally suited for investigating principles of archaeal transcription through analysis of the capacity to utilize a diversity of substrates for growth and methanogenesis. Methanosarcina species switch pathways in response to the most energetically favorable substrate, metabolizing methylotrophic substrates in preference to acetate marked by substantial regulation of gene expression. Although conversion of the methyl group of acetate accounts for most of the methane produced in Earth's biosphere, no proteins involved in the regulation of genes in the acetate pathway have been reported. The results presented here establish that MreA participates in the global regulation of diverse methanogenic pathways in the genus Methanosarcina. Finally, the results contribute to a broader understanding of transcriptional regulation in the domain Archaea.

Role of the fused corrinoid/methyl transfer protein CmtA during CO-dependent growth of Methanosarcina acetivorans.

The genome of Methanosarcina acetivorans encodes three homologs, initially annotated as hypothetical fused corrinoid/methyl transfer proteins, which are highly elevated in CO-grown cells versus cells grown with alternate substrates. Based only on phenotypic analyses of deletion mutants, it was previously concluded that the homologs are strictly dimethylsulfide:coenzyme M (CoM) methyltransferases not involved in the metabolism of CO (E. Oelgeschlager and M. Rother, Mol. Microbiol. 72:1260 -1272, 2009). The homolog encoded by MA4383 (here designated CmtA) was reexamined via biochemical characterization of the protein overproduced in Escherichia coli. Purified CmtA reconstituted with methylcob(III)alamin contained a molar ratio of cobalt to protein of 1.0 ± 0.2. The UV-visible spectrum was typical of methylated corrinoid-containing proteins, with absorbance maxima at 370 and 420 nm and a band of broad absorbance between 450 and 600 nm with maxima at 525, 490, and 550 nm. CmtA reconstituted with aquocobalamin showed methyl-tetrahydromethanopterin:CoM (CH(3)-THMPT:HS-CoM) methyltransferase activity (0.31 µmol/min/mg) with apparent K(m) values of 135 µM for CH(3)-THMPT and 277 µM for HS-CoM. The ratio of CH(3)-THMPT:HS-CoM methyltransferase activity in the soluble versus membrane cellular fractions was 15-fold greater in CO-grown versus methanol-grown cells. A mutant strain deleted for the CmtA gene showed lower growth rates and final yields when cultured with growth-limiting partial pressures of CO, demonstrating a role for CmtA during growth with this substrate. The results establish that CmtA is a soluble CH(3)-THSPT:HS-CoM methyltransferase postulated to supplement the membrane-bound CH(3)-THMPT:HS-CoM methyltransferase during CO-dependent growth of M. acetivorans. Thus, we propose that the name of the enzyme encoded by MA4384 be CmtA (for cytoplasmic methyltransferase).

Role of dipicolinic acid in the germination, stability, and viability of spores of Bacillus subtilis.

Spores of Bacillus subtilis spoVF strains that cannot synthesize dipicolinic acid (DPA) but take it up during sporulation were prepared in medium with various DPA concentrations, and the germination and viability of these spores as well as the DPA content in individual spores were measured. Levels of some other small molecules in DPA-less spores were also measured. These studies have allowed the following conclusions. (i) Spores with no DPA or low DPA levels that lack either the cortex-lytic enzyme (CLE) SleB or the receptors that respond to nutrient germinants could be isolated but were unstable and spontaneously initiated early steps in spore germination. (ii) Spores that lacked SleB and nutrient germinant receptors and also had low DPA levels were more stable. (iii) Spontaneous germination of spores with no DPA or low DPA levels was at least in part via activation of SleB. (iv) The other redundant CLE, CwlJ, was activated only by the release of high levels of DPA from spores. (v) Low levels of DPA were sufficient for the viability of spores that lacked most alpha/beta-type small, acid-soluble spore proteins. (vi) DPA levels accumulated in spores prepared in low-DPA-containing media varied greatly between individual spores, in contrast to the presence of more homogeneous DPA levels in individual spores made in media with high DPA concentrations. (vii) At least the great majority of spores of several spoVF strains that contained no DPA also lacked other major spore small molecules and had gone through some of the early reactions in spore germination.

Analysis of interactions between nutrient germinant receptors and SpoVA proteins of Bacillus subtilis spores.

Yeast two-hybrid and Far Western analyses were used to detect interactions between Bacillus subtilis spores' nutrient germinant receptor proteins and proteins encoded by the spoVA operon, all of which are involved in spore germination and located in the spores' inner membrane. These analyses indicated that two subunits of the GerA nutrient germinant receptor interact, consistent with previous genetic data, and that some GerA proteins interact with SpoVAD and some with SpoVAE. SpoVA proteins appear to be involved in the release of the spore's dipicolinic acid during spore germination, an event triggered by the binding of nutrient germinants to their receptors. Consequently, these new findings suggest that nutrient germinant receptors physically contact SpoVA proteins, and presumably this is a route for signal transduction during spore germination.

Levels of Ca2+-dipicolinic acid in individual bacillus spores determined using microfluidic Raman tweezers.

Pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) in a 1:1 chelate with calcium ion (Ca-DPA) comprises 5 to 15% of the dry weight of spores of Bacillus species. Ca-DPA is important in spore resistance to many environmental stresses and in spore stability, and Ca-DPA levels in spore populations can vary with spore species/strains, as well as with sporulation conditions. We have measured levels of Ca-DPA in large numbers of individual spores in populations of a variety of Bacillus species and strains by using microfluidic Raman tweezers, in which a single spore is trapped in a focused laser beam and its Ca-DPA is quantitated from the intensity of the Ca-DPA-specific band at 1,017 cm(-1) in Raman spectroscopy. Conclusions from these measurements include the following: (i) Ca-DPA concentrations in the spore core are >800 mM, well above Ca-DPA solubility; (ii) SpoVA proteins may be involved in Ca-DPA uptake in sporulation; and (iii) Ca-DPA levels differ significantly among individual spores in a population, but much of this variation could be due to variations in the sizes of individual spores.

Role of SpoVA proteins in release of dipicolinic acid during germination of Bacillus subtilis spores triggered by dodecylamine or lysozyme.

The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of approximately 9 and a temperature optimum of 60 degrees C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl(2). Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca(2+) almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca(2+) and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca(2+)-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca(2+)-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.

Localization of SpoVAD to the inner membrane of spores of Bacillus subtilis.

The products of the hexacistronic spoVA operon of Bacillus subtilis may be involved in the transport of dipicolinic acid into the forespore during sporulation and its release during spore germination. The major hydrophilic coding region of B. subtilis spoVAD was cloned, the protein was expressed in Escherichia coli as a His tag fusion protein, and a rabbit antiserum was raised against the purified protein. Western blot analyses of fractions from B. subtilis spores showed that SpoVAD is an integral inner membrane protein present at levels >50-fold higher than those of the spore's nutrient germinant receptors that are also present in the inner membrane. SpoVAD also persisted in outgrowing spores.

Analysis of the germination of spores of Bacillus subtilis with temperature sensitive spo mutations in the spoVA operon.

A Bacillus subtilis strain with a base substitution in the ribosome-binding site of spoVAC was temperature sensitive (ts) in sporulation and spores prepared at the permissive temperature were ts in L-alanine-triggered germination, but not in germination with Ca2+-dipicolinic acid (DPA) or dodecylamine. Spores of a ts spo mutant with a missense mutation in the spoVAC coding region were not ts for germination with l-alanine, dodecylamine or Ca2+-DPA. These findings are discussed in light of the proposal that SpoVA proteins are involved not only in DPA uptake during sporulation, but also in DPA release during nutrient-mediated spore germination.

Effects of overexpression of nutrient receptors on germination of spores of Bacillus subtilis.

The rates of germination of Bacillus subtilis spores with L-alanine were increased markedly, in particular at low L-alanine concentrations, by overexpression of the tricistronic gerA operon that encodes the spore's germinant receptor for L-alanine but not by overexpression of gerA operon homologs encoding receptors for other germinants. However, spores with elevated levels of the GerA proteins did not germinate more rapidly in a mixture of asparagine, glucose, fructose, and K(+) (AGFK), a germinant combination that requires the participation of at least the germinant receptors encoded by the tricistronic gerB and gerK operons. Overexpression of the gerB or gerK operon or both the gerB and gerK operons also did not stimulate spore germination in AGFK. Overexpression of a mutant gerB operon, termed gerB*, that encodes a receptor allowing spore germination in response to either D-alanine or L-asparagine also caused faster spore germination with these germinants, again with the largest enhancement of spore germination rates at lower germinant concentrations. However, the magnitudes of the increases in the germination rates with D-alanine or L-asparagine in spores overexpressing gerB* were well below the increases in the spore's levels of the GerBA protein. Germination of gerB* spores with D-alanine or L-asparagine did not require participation of the products of the gerK operon, but germination with these agents was decreased markedly in spores also overexpressing gerA. These findings suggest that (i) increases in the levels of germinant receptors that respond to single germinants can increase spore germination rates significantly; (ii) there is some maximum rate of spore germination above which stimulation of GerA operon receptors alone will not further increase the rate of spore germination, as action of some protein other than the germinant receptors can become rate limiting; (iii) while previous work has shown that the wild-type GerB and GerK receptors interact in some fashion to cause spore germination in AGFK, there also appears to be an additional component required for AGFK-triggered spore germination; (iv) activation of the GerB receptor with D-alanine or L-asparagine can trigger spore germination independently of the GerK receptor; and (v) it is likely that the different germinant receptors interact directly and/or compete with each other for some additional component needed for initiation of spore germination. We also found that very high levels of overexpression of the gerA or gerK operon (but not the gerB or gerB* operon) in the forespore blocked sporulation shortly after the engulfment stage, although sporulation appeared normal with the lower levels of gerA or gerK overexpression that were used to generate spores for analysis of rates of germination.