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Alchemical free energy perturbation (FEP) is a rigorous and powerful technique to calculate the free energy difference between distinct chemical systems. Here we report our implementation of automated large-scale FEP calculations, using the Amber software package, to facilitate antibody design and evaluation. In combination with Hamiltonian replica exchange, our FEP simulations aim to predict the effect of mutations on both the binding affinity and the structural stability. Importantly, we incorporate multiple strategies to faithfully estimate the statistical uncertainties in the FEP results. As a case study, we apply our protocols to systematically evaluate variants of the m396 antibody for their conformational stability and their binding affinity to the spike proteins of SARS-CoV-1 and SARS-CoV-2. By properly adjusting relevant parameters, the particle collapse problems in the FEP simulations are avoided. Furthermore, large statistical errors in a small fraction of the FEP calculations are effectively reduced by extending the sampling, such that acceptable statistical uncertainties are achieved for the vast majority of the cases with a modest total computational cost. Finally, our predicted conformational stability for the m396 variants is qualitatively consistent with the experimentally measured melting temperatures. Our work thus demonstrates the applicability of FEP in computational antibody design. [ABSTRACT FROM AUTHOR]

Rift Valley fever virus (RVFV) is a highly pathogenic mosquito-borne virus capable of causing hepatitis, encephalitis, blindness, hemorrhagic syndrome, and death in humans and livestock. Upon aerosol infection with RVFV, the brain is a major site of viral replication and tissue damage, yet pathogenesis in this organ has been understudied. Here, we investigated the immune response in the brain of RVFV infected mice. In response to infection, microglia initiated robust transcriptional upregulation of antiviral immune genes, as well as increased levels of activation markers and cytokine secretion that is dependent on mitochondrial antiviral-signaling protein (MAVS) and independent of toll-like receptors 3 and 7. In vivo, Mavs-/- mice displayed enhanced susceptibility to RVFV as determined by increased brain viral burden and higher mortality. Single-cell RNA sequence analysis identified defects in type I interferon and interferon responsive gene expression within microglia in Mavs-/- mice, as well as dysregulated lymphocyte infiltration. The results of this study provide a crucial step towards understanding the precise molecular mechanisms by which RVFV infection is controlled in the brain and will help inform the development of vaccines and antiviral therapies that are effective in preventing encephalitis. Author summary: Rift Valley fever virus causes severe disease in humans and livestock and in some cases can be fatal. There is concern about the use of Rift Valley fever virus as a bioweapon since it can be transmitted through the air, and there are no vaccines or antiviral treatments. Airborne transmission of the virus causes severe inflammation of the brain, yet little is known about the immune response against the virus in this organ. Here, we investigated the immune response in the brain to Rift Valley fever virus following intranasal infection. We determined that microglia, the resident immune cells of the brain, initiate a robust response to Rift Valley fever virus infection and identified a key immune pathway that is critical for the ability of microglia to respond to infection. When this immune pathway is rendered non-functional, mice have a dysregulated response to infection in the brain. This study provides insight into how the immune response can control Rift Valley fever virus infection of the brain. [ABSTRACT FROM AUTHOR]

Background: Recombinant expression of toxic proteins remains a challenging problem. One potential method to shield toxicity and thus improve expression of these proteins is to encapsulate them within protein compartments to sequester them away from their targets. Many bacteria naturally produce so-called bacterial microcompartments (BMCs) in which enzymes comprising a biosynthetic pathway are encapsulated in a proteinaeous shell, which is in part thought to shield the cells from the toxicity of reaction intermediates. As a proof-of-concept, we attempted to encapsulate toxic, lysis protein E (E) from bacteriophage ϕX174 inside recombinant BMCs to enhance its expression and achieve higher yields during downstream purification. Results: E was fused with various N-terminal BMC targeting tags (PduP-, PduD-, and EutC-tags, 18-20 amino acids) and co-expressed with appropriate BMC shell proteins that associate with the tags and are required to form BMCs. Only BMC targeted E fusions, but not non-tagged E, could be successfully cloned, suggesting that the BMC tags reduce the toxicity of E. A PduP-tagged E system appeared to achieve the highest expression of E. Co-expression of Pdu BMC shell proteins with PduP-E increased its expression by 20-50%. Affinity purification of PduP-E via Ni-NTA in the presence of Empigen BB detergent yielded 270 μg of PduP-E per L of induced culture. Removal of the PduP-tag via proteolysis resulted in a final yield of 200 μg of E per L of induced culture, a nearly order of magnitude (~sevenfold) improvement compared to prior reports. Conclusions: These results demonstrate improved expression of ϕX174 lysis protein E via re-directed BMC systems and ultimately higher E purification yields. Similar strategies can be used to enhance expression of other toxic proteins in recombinant Escherichia coli systems. [ABSTRACT FROM AUTHOR]

Background: Chlamydia is the most common sexually transmitted infection reported to the CDC, with over 1,500,000 cases reported in 2020 alone, yet formulation of a protective chlamydia vaccine remains elusive. In humans, chlamydia is caused by infection with the gram-negative bacteria Chlamydia tracomatis (Ct). Amino acid sequence diversity of Ct strains classify Ct into 15 distinct serovars. Human infections have been found to be a heterologous mix of these serovars, with serovar E being the most prevalent. The porin protein, major outer membrane protein (MOMP), is the most abundant protein in all Ct life cycle stages, making it a desirable vaccine target. Methods and Results: To determine the feasibility of a cross-serovar vaccine targeting MOMP, we performed a multi-protein sequence alignment of MOMP from all Ct serovars. As expected, the serovars are on average ~88% homologous to each other with majority of the sequence deviations concentrated within the previously described variable domain regions. To develop a cross-serovar vaccine against Ct, we then systematically analyzed the Immune Epitope DataBase (IEDB) to identify experimentally tested B- and T-cell epitopes against the aligned serovar E MOMP sequence. This computational analysis identified 11 linear sequences as putative epitope candidates. Of these candidates, 5 were predicted to bind T-cells, 2 were predicted to bind B-cells, and 4 were predicted to bind both immune cell types. To test these epitopes for desirable vaccine qualities, such as self-serovar/ cross-serovar immunogenicity and specific CD8+ T-cell responses, we tested 14 antigen permutations containing 1-8 of the 11 putative epitopes in various immunological-based assays. Conclusions: Through systemic analysis of established experiential data, we were able to identify 11 putative epitope sequences targeting Ct MOMP. We screened all 11 epitopes for single and synergistic effects promoting (or deterring) the desired qualities of a Ct vaccine, such as self-serovar and cross-serovar immunogenicity. Future iterations of the multi-epitope antigen identified in these studies can be used for development of either protein subunit or RNA-based Ct vaccines. [ABSTRACT FROM AUTHOR]

Lytic proteins encoded by bacterial genomes have been implicated in cell wall biosynthesis and recycling. The Bacillus cereus E33L ampD gene encodes a putative N-acetylmuramoyl-L-alanine amidase. This gene, expressed in vitro, produced a very stable, highly active lytic protein. Very low concentrations rapidly and efficiently lyse vegetative Bacillus anthracis cells. [ABSTRACT FROM AUTHOR]

Fluorescence correlation spectroscopy(FCS) is an important techniquefor studying biochemical interactions dynamically that may be used in vitroand in cell-based studies. It is generally claimedthat FCS may only be used at nM concentrations. We show that thisgeneral consensus is incorrect and that the limitation to nM concentrationsis not fundamental but due to detector limits as well as laser fluctuations.With a high count rate detector system and applying laser fluctuationcorrections, we demonstrate FCS measurements up to 38 μM withthe same signal-to-noise as at lower concentrations. Optical nanoconfinementapproaches previously used to increase the concentration range ofFCS are not necessary, and further increases above 38 μM maybe expected using detectors and detector arrays with higher saturationrates and better laser fluctuation corrections. This approach greatlywidens the possibilities of dynamic measurements of biochemical interactionsusing FCS at physiological concentrations. [ABSTRACT FROM AUTHOR]

Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Longterm infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, and protein misfolding. Here, we used an Escherichia coli-based cell-free system to express aMOMPprotein from the mouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP-tNLP). The cell-free expressed mMOMP-tNLPs contain mMOMP multimers similar to the nativeMOMPprotein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP-tNLP complex in a 1-ml cell-free reaction. The mMOMP-tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP-tNLP formulation, we demonstrate a unique approach to solubilizing and administering membranebound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity. [ABSTRACT FROM AUTHOR]

Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, provide better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. Genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms. [ABSTRACT FROM AUTHOR]

Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteins as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. These studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses. [ABSTRACT FROM AUTHOR]