Identification of an Optimized Receptor-Binding Domain Subunit Vaccine against SARS-CoV-2.

Current vaccine efforts to combat SARS-CoV-2 are focused on the whole spike protein administered as mRNA, viral vector, or protein subunit. However, the SARS-CoV-2 receptor-binding domain (RBD) is the immunodominant portion of the spike protein, accounting for 90% of serum neutralizing activity. In this study, we constructed several versions of RBD and together with aluminum hydroxide or DDA (dimethyldioctadecylammonium bromide)/TDB (d-(+)-trehalose 6,6'-dibehenate) adjuvant evaluated immunogenicity in mice. We generated human angiotensin-converting enzyme 2 knock-in mice to evaluate vaccine efficacy in vivo following viral challenge. We found that 1) subdomain (SD)1 was essential for the RBD to elicit maximal immunogenicity; 2) RBDSD1 produced in mammalian HEK cells elicited better immunogenicity than did protein produced in insect or yeast cells; 3) RBDSD1 combined with the CD4 Th1 adjuvant DDA/TDB produced higher neutralizing Ab responses and stronger CD4 T cell responses than did aluminum hydroxide; 4) addition of monomeric human Fc receptor to RBDSD1 (RBDSD1Fc) significantly enhanced immunogenicity and neutralizing Ab titers; 5) the Beta version of RBDSD1Fc provided a broad range of cross-neutralization to multiple antigenic variants of concern, including Omicron; and 6) the Beta version of RBDSD1Fc with DDA/TDB provided complete protection against virus challenge in the knock-in mouse model. Thus, we have identified an optimized RBD-based subunit vaccine suitable for clinical trials.

Reverse vaccinology-based identification of a novel surface lipoprotein that is an effective vaccine antigen against bovine infections caused by Pasteurella multocida.

Pasteurella multocida can infect a multitude of wild and domesticated animals, with infections in cattle resulting in hemorrhagic septicemia (HS) or contributing to bovine respiratory disease (BRD) complex. Current cattle vaccines against P. multocida consist of inactivated bacteria, which only offer limited and serogroup specific protection. Here, we describe a newly identified surface lipoprotein, PmSLP, that is present in nearly all annotated P. multocida strains isolated from cattle. Bovine associated variants span three of the four identified phylogenetic clusters, with PmSLP-1 and PmSLP-2 being restricted to BRD associated isolates and PmSLP-3 being restricted to isolates associated with HS. Recombinantly expressed, soluble PmSLP-1 (BRD-PmSLP) and PmSLP-3 (HS-PmSLP) vaccines were both able to provide full protection in a mouse sepsis model against the matched P. multocida strain, however no cross-protection and minimal serum IgG cross-reactivity was identified. Full protection against both challenge strains was achieved with a bivalent vaccine containing both BRD-PmSLP and HS-PmSLP, with serum IgG from immunized mice being highly reactive to both variants. Year-long stability studies with lyophilized antigen stored under various temperatures show no appreciable difference in biophysical properties or loss of efficacy in the mouse challenge model. PmSLP-1 and PmSLP-3 vaccines were each evaluated for immunogenicity in two independent cattle trials involving animals of different age ranges and breeds. In all four trials, vaccination with PmSLP resulted in an increase in antigen specific serum IgG over baseline. In a blinded cattle challenge study with a recently isolated HS strain, the matched HS-PmSLP vaccine showed strong efficacy (75-87.5% survival compared to 0% in the control group). Together, these data suggest that cattle vaccines composed of PmSLP antigens can be a practical and effective solution for preventing HS and BRD related P. multocida infections.

Preclinical evaluation of a SARS-CoV-2 mRNA vaccine PTX-COVID19-B.

Safe and effective vaccines are needed to end the COVID-19 pandemic. Here, we report the preclinical development of a lipid nanoparticle­formulated SARS-CoV-2 mRNA vaccine, PTX-COVID19-B. PTX-COVID19-B was chosen among three candidates after the initial mouse vaccination results showed that it elicited the strongest neutralizing antibody response against SARS-CoV-2. Further tests in mice and hamsters indicated that PTX-COVID19-B induced robust humoral and cellular immune responses and completely protected the vaccinated animals from SARS-CoV-2 infection in the lung. Studies in hamsters also showed that PTX-COVID19-B protected the upper respiratory tract from SARS-CoV-2 infection. Mouse immune sera elicited by PTX-COVID19-B vaccination were able to neutralize SARS-CoV-2 variants of concern, including the Alpha, Beta, Gamma, and Delta lineages. No adverse effects were induced by PTX-COVID19-B in either mice or hamsters. Based on these results, PTX-COVID19-B was authorized by Health Canada to enter clinical trials in December 2020 with a phase 2 clinical trial ongoing.

Intranasal HD-Ad vaccine protects the upper and lower respiratory tracts of hACE2 mice against SARS-CoV-2.

BACKGROUND: The ongoing COVID-19 pandemic has resulted in 185 million recorded cases and over 4 million deaths worldwide. Several COVID-19 vaccines have been approved for emergency use in humans and are being used in many countries. However, all the approved vaccines are administered by intramuscular injection and this may not prevent upper airway infection or viral transmission. RESULTS: Here, we describe a novel, intranasally delivered COVID-19 vaccine based on a helper-dependent adenoviral (HD-Ad) vector. The vaccine (HD-Ad_RBD) produces a soluble secreted form of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and we show it induced robust mucosal and systemic immunity. Moreover, intranasal immunization of K18-hACE2 mice with HD-Ad_RBD using a prime-boost regimen, resulted in complete protection of the upper respiratory tract against SARS-CoV-2 infection. CONCLUSION: Our approaches provide a powerful platform for constructing highly effective vaccines targeting SARS-CoV-2 and its emerging variants.

A Slam-dependent hemophore contributes to heme acquisition in the bacterial pathogen Acinetobacter baumannii.

Nutrient acquisition systems are often crucial for pathogen growth and survival during infection, and represent attractive therapeutic targets. Here, we study the protein machinery required for heme uptake in the opportunistic pathogen Acinetobacter baumannii. We show that the hemO locus, which includes a gene encoding the heme-degrading enzyme, is required for high-affinity heme acquisition from hemoglobin and serum albumin. The hemO locus includes a gene coding for a heme scavenger (HphA), which is secreted by a Slam protein. Furthermore, heme uptake is dependent on a TonB-dependent receptor (HphR), which is important for survival and/or dissemination into the vasculature in a mouse model of pulmonary infection. Our results indicate that A. baumannii uses a two-component receptor system for the acquisition of heme from host heme reservoirs.

Utility of Hybrid Transferrin Binding Protein Antigens for Protection Against Pathogenic Neisseria Species.

The surface transferrin receptor proteins from Neisseria gonorrhoeae have been recognized as ideal vaccine targets due to their critical role in survival in the human male genitourinary tract. Recombinant forms of the surface lipoprotein component of the receptor, transferrin binding protein B (TbpB), can be readily produced at high levels in the Escherichia coli cytoplasm and is suitable for commercial vaccine production. In contrast, the integral outer membrane protein, transferrin binding protein A (TbpA), is produced at relatively low levels in the outer membrane and requires detergents for solubilization and stabilization, processes not favorable for commercial applications. Capitalizing on the core ß-barrel structural feature common to the lipoprotein and integral outer membrane protein we engineered the lipoprotein as a scaffold for displaying conserved surface epitopes from TbpA. A stable version of the C-terminal domain of TbpB was prepared by replacing four larger exposed variable loops with short linking peptide regions. Four surface regions from the plug and barrel domains of Neisseria TbpA were transplanted onto this TbpB C-lobe scaffold, generating stable hybrid antigens. Antisera generated in mice and rabbits against the hybrid antigens recognized TbpA at the surface of Neisseria meningitidis and inhibited transferrin-dependent growth at levels comparable or better than antisera directed against the native TbpA protein. Two of the engineered hybrid antigens each elicited a TbpA-specific bactericidal antibody response comparable to that induced by TbpA. A hybrid antigen generated using a foreign scaffold (TbpB from the pig pathogen Haemophilus parasuis) displaying neisserial TbpA loop 10 was evaluated in a model of lower genital tract colonization by N. gonorrhoeae and a model of invasive infection by N. meningitidis. The loop 10 hybrid antigen was as effective as full length TbpA in eliminating N. gonorrhoeae from the lower genital tract of female mice and was protective against the low dose invasive infection by N. meningitidis. These results demonstrate that TbpB or its derivatives can serve as an effective scaffold for displaying surface epitopes of integral outer membrane antigens and these antigens can elicit protection against bacterial challenge.

Recombinant BCG Overexpressing phoP-phoR Confers Enhanced Protection against Tuberculosis.

The live tuberculosis vaccine Mycobacterium bovis BCG (Bacille Calmette-Guérin) comprises a number of genetically distinct substrains. In BCG-Prague, phoP of the PhoP-PhoR two-component system is a pseudogene due to a single insertion mutation. We hypothesized that this mutation partially accounts for the low immunogenicity of BCG-Prague observed in the 1970s. In this study, we showed that complementation with the M. bovis allele of phoP restored BCG-Prague's immunogenicity. Furthermore, we showed that overexpression of the M. bovis allele of phoP-phoR in BCG-Japan, a strain already containing a copy of phoP-phoR, further enhanced immunogenicity and protective efficacy. Vaccination of C57BL/6 mice with the recombinant strain rBCG-Japan/PhoPR induced higher levels of interferon-γ (IFN-γ) production by CD4+ T cells than that with the parental BCG. Guinea pigs vaccinated with rBCG-Japan/PhoPR were better protected against challenge with Mycobacterium tuberculosis than those immunized with the parental BCG, showing significantly longer survival time, reduced bacterial burdens, and less severe pathology. Taken together, our study has identified a genetic modification that could be generally applied to generate new recombinant BCG vaccines.

Transcription factors Rv0081 and Rv3334 connect the early and the enduring hypoxic response of Mycobacterium tuberculosis.

The ability of Mycobacterium tuberculosis (M. tb) to survive and persist in the host for decades in an asymptomatic state is an important aspect of tuberculosis pathogenesis. Although adaptation to hypoxia is thought to play a prominent role underlying M. tb persistence, how the bacteria achieve this goal is largely unknown. Rv0081, a member of the DosR regulon, is induced at the early stage of hypoxia while Rv3334 is one of the enduring hypoxic response genes. In this study, we uncovered genetic interactions between these two transcription factors. RNA-seq analysis of ΔRv0081 and ΔRv3334 revealed that the gene expression profiles of these two mutants were highly similar. We also found that under hypoxia, Rv0081 positively regulated the expression of Rv3334 while Rv3334 repressed transcription of Rv0081. In addition, we demonstrated that Rv0081 formed dimer and bound to the promoter region of Rv3334. Taken together, these data suggest that Rv0081 and Rv3334 work in the same regulatory pathway and that Rv3334 functions immediately downstream of Rv0081. We also found that Rv3334 is a bona fide regulator of the enduring hypoxic response genes. Our study has uncovered a regulatory pathway that connects the early and the enduring hypoxic response, revealing a transcriptional cascade that coordinates the temporal response of M. tb to hypoxia.

Humane Endpoints for Guinea Pigs Used for Mycobacterium tuberculosis Vaccine Research.

Guinea pigs are a commonly used model for tuberculosis vaccine research. Loss of body weight is the most frequently described humane endpoint for animals used in these studies. During a chronic study, we noted labored breathing in some tuberculosis-infected guinea pigs. To develop consistent humane endpoints for these guinea pigs, we performed an observational study using multiple clinical signs. A combination of body weight loss, labored breathing, and activity level during handling estimated the time to euthanasia within approximately 7 d. Histologic severity scores of lesions in the cranial or caudal lung lobe (or both) supported clinical endpoints. This study presents humane endpoints for the refinement of studies using guinea pigs in tuberculosis research.

Loss of Lipid Virulence Factors Reduces the Efficacy of the BCG Vaccine.

Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the only vaccine available for tuberculosis (TB) control. BCG comprises a number of substrains that exhibit genetic and biochemical differences. Whether and how these differences affect BCG efficacy remain unknown. Compared to other BCG strains, BCG-Japan, -Moreau, and -Glaxo are defective in the production of phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs), two lipid virulence factors. To determine if the loss of PDIMs/PGLs affects BCG efficacy, we constructed a PDIM/PGL-deficient strain of BCG-Pasteur by deleting fadD28, and compared virulence, immunogenicity, and protective efficacy in animal models. SCID mouse infection experiments showed that ∆fadD28 was more attenuated than wild type (WT). The ∆fadD28 and WT strains induced equivalent levels of antigen specific IFN-γ by CD4(+) and CD8(+) T cells; however, ∆fadD28 was less effective against Mycobacterium tuberculosis challenge in both BALB/c mice and guinea pigs. These results indicate that the loss of PIDMs/PGLs reduces the virulence and protective efficacy of BCG. Since the loss of PDIMs/PGLs occurs naturally in a subset of BCG strains, it also suggests that these strains may have been over-attenuated, which compromises their effectiveness. Our finding has important implications for current BCG programs and future vaccine development.

Deglycosylation as a mechanism of inducible antibiotic resistance revealed using a global relational tree for one-component regulators.

The ligands that interact with the vast majority of small-molecule binding transcription factors are unknown, a significant gap in our understanding of sensory perception by cells. TetR-family regulators (TFRs) are found in most prokaryotes and are involved in regulating virtually every aspect of prokaryotic life however only a few TFRs have been characterized. We report the application of phylogenomics to the identification of cognate ligands for TFRs. Using phylogenomics we identify a TFR, KijR, that responds to the antibiotic kijanimicin. We go on to show that KijR represses a gene, kijX, which confers resistance to kijanimicin. Finally we show that KijX inactivates kijanimicin by the hydrolytic removal of sugar residues. This is a demonstration of antibiotic resistance by deglycosylation.

Genome context as a predictive tool for identifying regulatory targets of the TetR family transcriptional regulators.

TetR family transcriptional regulators (TFRs) are found in most bacteria and archea. Most of the family members that have been investigated to date are repressors of their target genes, and the majority of these, like the well-characterized protein TetR, regulate genes that encode transmembrane efflux pumps. In many cases repression by TFR proteins is reversed through the direct binding of a small-molecule ligand. The number of TFRs in the public database has grown rapidly as a result of genome sequencing and there are now thousands of family members; however virtually nothing is known about the biology and biochemistry they regulate. Generally applicable methods for predicting their regulatory targets would assist efforts to characterize the family. Here, we investigate chromosomal context of 372 TFRs from three Streptomyces species. We find that the majority (250 TFRs) are transcribed divergently from one neighboring gene, as is the case for TetR and its target tetA. We explore predicted target gene product identity and intergenic separation to see which either correlates with a direct regulatory relationship. While intergenic separation is a critical factor in regulatory prediction the identity of the putative target gene product is not. Our data suggest that those TFRs that are <200 bp from their divergently oriented neighbors are most likely to regulate them. These target genes include membrane proteins (26% of which 22% are probable membrane-associated pumps), enzymes (60%), other proteins such as transcriptional regulators (1%), and proteins having no predictive sequence motifs (13%). In addition to establishing a solid foundation for identifying targets for TFRs of unknown function, our analysis demonstrates a much greater diversity of TFR-regulated biochemical functions.

Coupling of the biosynthesis and export of the DNA gyrase inhibitor simocyclinone in Streptomyces antibioticus.

Because most antibiotics are potentially lethal to the producing organism, there must be mechanisms to ensure that the machinery responsible for export of the mature antibiotic is in place at the time of biosynthesis. Simocyclinone D8 is a potent DNA gyrase inhibitor produced by Streptomyces antibioticus Tü 6040. Within the simocyclinone biosynthetic cluster are two divergently transcribed genes, simR and simX, encoding proteins that resemble the TetR/TetA repressor-efflux pump pair that cause widespread resistance to clinically important tetracyclines. Engineered expression of simX from a strong, heterologous promoter conferred high level simocyclinone D8 resistance on Streptomyces lividans, showing that simX encodes a simocyclinone efflux pump. Transcription of simX is controlled by SimR, which directly represses the simX and simR promoters by binding to two operator sites in the simX-simR intergenic region. Simocyclinone D8 abolishes DNA binding by SimR, providing a mechanism that couples the biosynthesis of simocyclinone to its export. In addition, an intermediate in the biosynthetic pathway, simocyclinone C4, which is essentially inactive as a DNA gyrase inhibitor, also induces simX expression in vivo and relieves simX repression by SimR in vitro.

Investigation of transcription repression and small-molecule responsiveness by TetR-like transcription factors using a heterologous Escherichia coli-based assay.

The SCO7222 protein and ActR are two of approximately 150 TetR-like transcription factors encoded in the Streptomyces coelicolor genome. Using bioluminescence as a readout, we have developed Escherichia coli-based biosensors that accurately report the regulatory activity of these proteins and used it to investigate their interactions with DNA and small-molecule ligands. We found that the SCO7222 protein and ActR repress the expression of their putative target genes, SCO7223 and actII-ORF2 (actA), respectively, by interacting with operator sequence in the promoters. The operators recognized by the two proteins are related such that O(7223) (an operator for SCO7223) could be bound by both the SCO7222 protein and ActR with similar affinities. In contrast, O(act) (an operator for actII-ORF2) was bound tightly by ActR and more weakly by the SCO7222 protein. We demonstrated ligand specificity of these proteins by showing that while TetR (but not ActR or the SCO7222 protein) interacts with tetracyclines, ActR (but not TetR or the SCO7222 protein) interacts with actinorhodin and related molecules. Through operator-targeted mutagenesis, we found that at least two nucleotide changes in O(7223) were required to disrupt its interaction with SCO7222 protein, while ActR was more sensitive to changes on O(act). Most importantly, we found that the interaction of each protein with wild-type and mutant operator sequences in vivo and in vitro correlated perfectly. Our data suggest that E. coli-based biosensors of this type should be broadly applicable to TetR-like transcription factors.

Initiation of actinorhodin export in Streptomyces coelicolor.

Many microorganisms produce molecules having antibiotic activity and expel them into the environment, presumably enhancing their ability to compete with their neighbours. Given that these molecules are often toxic to the producer, mechanisms must exist to ensure that the assembly of the export apparatus accompanies or precedes biosynthesis. Streptomyces coelicolor produces the polyketide antibiotic actinorhodin in a multistep pathway involving enzymes encoded by genes that are clustered together. Embedded within the cluster are genes for actinorhodin export, two of which, actR and actA resemble the classic tetR and tetA repressor/efflux pump-encoding gene pairs that confer resistance to tetracycline. Like TetR, which represses tetA, ActR is a repressor of actA. We have identified several molecules that can relieve repression by ActR. Importantly (S)-DNPA (an intermediate in the actinorhodin biosynthetic pathway) and kalafungin (a molecule related to the intermediate dihydrokalafungin), are especially potent ActR ligands. This suggests that along with the mature antibiotic(s), intermediates in the biosynthetic pathway might activate expression of the export genes thereby coupling export to biosynthesis. We suggest that this could be a common feature in the production of many bioactive natural products.