Pharmacokinetic and pharmacodynamic analysis of baloxavir marboxil, a novel cap-dependent endonuclease inhibitor, in a murine model of influenza virus infection.

BACKGROUND: Baloxavir acid, the active form of the orally available prodrug baloxavir marboxil, is a novel cap-dependent endonuclease inhibitor of influenza virus. Baloxavir marboxil has been shown to rapidly reduce virus titres compared with oseltamivir in clinical studies. OBJECTIVES: We investigated the relationship between pharmacokinetic (PK) parameters and antiviral activity of baloxavir acid based on virus titre reduction in lungs of infected mice. METHODS: BALB/c mice infected with a sub-lethal dose of influenza A(H1N1), A(H1N1)pdm09, A(H3N2) or type B virus were treated on day 5 with oral baloxavir marboxil (0.5-50 mg/kg q12h), subcutaneous baloxavir acid (0.25-8 mg/kg/day), oseltamivir phosphate (5 or 50 eq mg/kg q12h) or other antivirals for 1 day. Lung virus titres were assessed 24 h after initial antiviral dosing. PK testing was performed at up to 24 h post-dosing of baloxavir marboxil or baloxavir acid in A/WSN/33-infected mice and the PK/pharmacodynamic (PD) relationship was evaluated for baloxavir acid. RESULTS: Oral baloxavir marboxil administration showed dose-dependent virus titre reductions in lungs of mice infected with the different types/subtypes of influenza viruses 24 h post-dosing. Baloxavir marboxil at 15 mg/kg q12h resulted in ≥100-fold and ≥10-fold reductions in influenza A and B virus titres, respectively, compared with oseltamivir phosphate. PK/PD analysis showed that the plasma concentration at the end of the dosing interval (Cτ) or the plasma concentration at 24 h after initial dosing (C24) was the PK parameter predicting the virus titres at 24 h post-dosing of baloxavir acid. CONCLUSIONS: PK/PD analysis of baloxavir acid based on virus titre reduction in this mouse model could be helpful in predicting and maximizing virological outcomes in clinical settings.

B cell-intrinsic MyD88 signaling controls IFN-γ-mediated early IgG2c class switching in mice in response to a particulate adjuvant.

Adjuvants improve the potency of vaccines, but the modes of action (MOAs) of most adjuvants are largely unknown. TLR-dependent and -independent innate immune signaling through the adaptor molecule MyD88 has been shown to be pivotal to the effects of most adjuvants; however, MyD88's involvement in the TLR-independent MOAs of adjuvants is poorly understood. Here, using the T-dependent antigen NIPOVA and a unique particulate adjuvant called synthetic hemozoin (sHZ), we show that MyD88 is required for early GC formation and enhanced antibody class-switch recombination (CSR) in mice. Using cell-type-specific MyD88 KO mice, we found that IgG2c class switching, but not IgG1 class switching, was controlled by B cell-intrinsic MyD88 signaling. Notably, IFN-γ produced by various cells including T cells, NK cells, and dendritic cells was the primary cytokine for IgG2c CSR and B-cell intrinsic MyD88 is required for IFN-γ production. Moreover, IFN-γ receptor (IFNγR) deficiency abolished sHZ-induced IgG2c production, while recombinant IFN-γ administration successfully rescued IgG2c CSR impairment in mice lacking B-cell intrinsic MyD88. Together, our results show that B cell-intrinsic MyD88 signaling is involved in the MOA of certain particulate adjuvants and this may enhance our specific understanding of how adjuvants and vaccines work.

Hydroxypropyl-ß-cyclodextrin spikes local inflammation that induces Th2 cell and T follicular helper cell responses to the coadministered antigen.

Cyclodextrins are commonly used as a safe excipient to enhance the solubility and bioavailability of hydrophobic pharmaceutical agents. Their efficacies and mechanisms as drug-delivery systems have been investigated for decades, but their immunological properties have not been examined. In this study, we reprofiled hydroxypropyl-ß-cyclodextrin (HP-ß-CD) as a vaccine adjuvant and found that it acts as a potent and unique adjuvant. HP-ß-CD triggered the innate immune response at the injection site, was trapped by MARCO(+) macrophages, increased Ag uptake by dendritic cells, and facilitated the generation of T follicular helper cells in the draining lymph nodes. It significantly enhanced Ag-specific Th2 and IgG Ab responses as potently as did the conventional adjuvant, aluminum salt (alum), whereas its ability to induce Ag-specific IgE was less than that of alum. At the injection site, HP-ß-CD induced the temporary release of host dsDNA, a damage-associated molecular pattern. DNase-treated mice, MyD88-deficient mice, and TBK1-deficient mice showed significantly reduced Ab responses after immunization with this adjuvant. Finally, we demonstrated that HP-ß-CD-adjuvanted influenza hemagglutinin split vaccine protected against a lethal challenge with a clinically isolated pandemic H1N1 influenza virus, and the adjuvant effect of HP-ß-CD was demonstrated in cynomolgus macaques. Our results suggest that HP-ß-CD acts as a potent MyD88- and TBK1-dependent T follicular helper cell adjuvant and is readily applicable to various vaccines.

Peptide-to-Small Molecule: Discovery of Non-Covalent, Active-Site Inhibitors of ß-Herpesvirus Proteases.

Viral proteases, the key enzymes that regulate viral replication and assembly, are promising targets for antiviral drug discovery. Herpesvirus proteases are enzymes with no crystallographically confirmed noncovalent active-site binders, owing to their shallow and polar substrate-binding pockets. Here, we applied our previously reported "Peptide-to-Small Molecule" strategy to generate novel inhibitors of ß-herpesvirus proteases. Rapid selection with a display technology was used to identify macrocyclic peptide 1 bound to the active site of human cytomegalovirus protease (HCMVPro) with high affinity, and pharmacophore queries were defined based on the results of subsequent intermolecular interaction analyses. Membrane-permeable small molecule 19, designed de novo according to this hypothesis, exhibited enzyme inhibitory activity (IC50 = 10-6 to 10-7 M) against ß-herpesvirus proteases, and the design concept was proved by X-ray cocrystal analysis.

A-910823, a squalene-based emulsion adjuvant, induces T follicular helper cells and humoral immune responses via α-tocopherol component.

Background: Adjuvants are chemical or biological materials that enhance the efficacy of vaccines. A-910823 is a squalene-based emulsion adjuvant used for S-268019-b, a novel vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is currently in clinical development. Published evidence has demonstrated that A-910823 can enhance the induction of neutralizing antibodies against SARS-CoV-2 in humans and animal models. However, the characteristics and mechanisms of the immune responses induced by A-910823 are not yet known. Methods and Results: To characterize A-910823, we compared the adaptive immune response profile enhanced by A-910823 with that of other adjuvants (AddaVax, QS21, aluminum salt-based adjuvants, and empty lipid nanoparticle [eLNP]) in a murine model. Compared with other adjuvants, A-910823 enhanced humoral immune responses to an equal or greater extent following potent T follicular helper (Tfh) and germinal center B (GCB) cell induction, without inducing a strong systemic inflammatory cytokine response. Furthermore, S-268019-b containing A-910823 adjuvant produced similar results even when given as a booster dose following primary administration of a lipid nanoparticle-encapsulated messenger RNA (mRNA-LNP) vaccine. Preparation of modified A-910823 adjuvants to identify which components of A-910823 play a role in driving the adjuvant effect and detailed evaluation of the immunological characteristics induced by each adjuvant showed that the induction of humoral immunity and Tfh and GCB cell induction in A-910823 were dependent on α-tocopherol. Finally, we revealed that the recruitment of inflammatory cells to the draining lymph nodes and induction of serum cytokines and chemokines by A-910823 were also dependent on the α-tocopherol component. Conclusions: This study demonstrates that the novel adjuvant A-910823 is capable of robust Tfh cell induction and humoral immune responses, even when given as a booster dose. The findings also emphasize that α-tocopherol drives the potent Tfh-inducing adjuvant function of A-910823. Overall, our data provide key information that may inform the future production of improved adjuvants.

Novel Adjuvant S-540956 Targets Lymph Nodes and Reduces Genital Recurrences and Vaginal Shedding of HSV-2 DNA When Administered with HSV-2 Glycoprotein D as a Therapeutic Vaccine in Guinea Pigs.

Herpes simplex virus type 2 (HSV-2) is a leading cause of genital ulcer disease and a major risk factor for acquisition and transmission of HIV. Frequent recurrent genital lesions and concerns about transmitting infection to intimate partners affect the quality of life of infected individuals. Therapeutic vaccines are urgently needed to reduce the frequency of genital lesions and transmission. S-540956 is a novel vaccine adjuvant that contains CpG oligonucleotide ODN2006 annealed to its complementary sequence and conjugated to a lipid that targets the adjuvant to lymph nodes. Our primary goal was to compare S-540956 administered with HSV-2 glycoprotein D (gD2) with no treatment in a guinea pig model of recurrent genital herpes (studies 1 and 2). Our secondary goals were to compare S-540956 with oligonucleotide ODN2006 (study1) or glucopyranosyl lipid A in a stable oil-in-water nano-emulsion (GLA-SE) (study 2). gD2/S-540956 reduced the number of days with recurrent genital lesions by 56%, vaginal shedding of HSV-2 DNA by 49%, and both combined by 54% compared to PBS, and was more efficacious than the two other adjuvants. Our results indicate that S-540956 has great potential as an adjuvant for a therapeutic vaccine for genital herpes, and merits further evaluation with the addition of potent T cell immunogens.

S-540956, a CpG Oligonucleotide Annealed to a Complementary Strand With an Amphiphilic Chain Unit, Acts as a Potent Cancer Vaccine Adjuvant by Targeting Draining Lymph Nodes.

Robust induction of cancer-antigen-specific CD8+ T cells is essential for the success of cancer peptide vaccines, which are composed of a peptide derived from a cancer-specific antigen and an immune-potentiating adjuvant, such as a Toll-like receptor (TLR) agonist. Efficient delivery of a vaccine antigen and an adjuvant to antigen-presenting cells in the draining lymph nodes (LNs) holds key to maximize vaccine efficacy. Here, we developed S-540956, a novel TLR9-agonistic adjuvant consisting of B-type CpG ODN2006 (also known as CpG7909), annealed to its complementary sequence oligodeoxynucleotide (ODN) conjugated to a lipid; it could target both a cancer peptide antigen and a CpG-adjuvant in the draining LNs. S-540956 accumulation in the draining LNs and activation of plasmacytoid dendritic cells (pDCs) were significantly higher than that of ODN2006. Mechanistic analysis revealed that S-540956 enhanced the induction of MHC class I peptide-specific CD8+ T cell responses via TLR9 in a CD4+ T cell-independent manner. In mice, the therapeutic effect of S-540956-adjuvanted with a human papillomavirus (HPV)-E7 peptide vaccine against HPV-E7-expressing TC-1 tumors was significantly better than that of an ODN2006-adjuvanted vaccine. Our findings demonstrate a novel adjuvant discovery with the complementary strand conjugated to a lipid, which enabled draining LN targeting and increased ODN2006 accumulation in draining LNs, thereby enhancing the adjuvant effect. Our findings imply that S-540956 is a promising adjuvant for cancer peptide vaccines and has a high potential for applications in various vaccines, including recombinant protein vaccines.

The role of RND-type efflux pumps in multidrug-resistant mutants of Klebsiella pneumoniae.

The emergence of multidrug-resistant Klebsiella pneumoniae is a worldwide problem. K. pneumoniae possesses numerous resistant genes in its genome. We isolated mutants resistant to various antimicrobials in vitro and investigated the importance of intrinsic genes in acquired resistance. The isolation frequency of the mutants was 10-7-10-9. Of the multidrug-resistant mutants, hyper-multidrug-resistant mutants (EB256-1, EB256-2, Nov1-8, Nov2-2, and OX128) were identified, and accelerated efflux activity of ethidium from the inside to the outside of the cells was observed in these mutants. Therefore, we hypothesized that the multidrug efflux pump, especially RND-type efflux pump, would be related to changes of the phenotype. We cloned all RND-type multidrug efflux pumps from the K. pneumoniae genome and characterized them. KexEF and KexC were powerful multidrug efflux pumps, in addition to AcrAB, KexD, OqxAB, and EefABC, which were reported previously. It was revealed that the expression of eefA was increased in EB256-1 and EB256-2: the expression of oqxA was increased in OX128; the expression of kexF was increased in Nov2-2. It was found that a region of 1,485 bp upstream of kexF, was deleted in the genome of Nov2-2. K. pneumoniae possesses more potent RND-multidrug efflux systems than E. coli. However, we revealed that most of them did not contribute to the drug resistance of our strain at basic levels of expression. On the other hand, it was also noted that the overexpression of these pumps could lead to multidrug resistance based on exposure to antimicrobial chemicals. We conclude that these pumps may have a role to maintain the intrinsic resistance of K. pneumoniae when they are overexpressed. The antimicrobial chemicals selected many resistant mutants at the same minimum inhibitory concentration (MIC) or a concentration slightly higher than the MIC. These results support the importance of using antibiotics at appropriate concentrations at clinical sites.

Characterization of MATE-type multidrug efflux pumps from Klebsiella pneumoniae MGH78578.

We previously described the cloning of genes related to drug resistance from Klebsiella pneumoniae MGH78578. Of these, we identified a putative gene encoding a MATE-type multidrug efflux pump, and named it ketM. Escherichia coli KAM32 possessing ketM on a plasmid showed increased minimum inhibitory concentrations for norfloxacin, ciprofloxacin, cefotaxime, acriflavine, Hoechst 33342, and 4',6-diamidino-2-phenyl indole (DAPI). The active efflux of DAPI was observed in E. coli KAM32 possessing ketM on a plasmid. The expression of mRNA for ketM was observed in K. pneumoniae cells, and we subsequently disrupted ketM in K. pneumoniae ATCC10031. However, no significant changes were observed in drug resistance levels between the parental strain ATCC10031 and ketM disruptant, SKYM. Therefore, we concluded that KetM was a multidrug efflux pump, that did not significantly contribute to intrinsic resistance to antimicrobial chemicals in K. pneumoniae. MATE-type transporters are considered to be secondary transporters; therefore, we investigated the coupling cations of KetM. DAPI efflux by KetM was observed when lactate was added to produce a proton motive force, indicating that KetM effluxed substrates using a proton motive force. However, the weak efflux of DAPI by KetM was also noted when NaCl was added to the assay mixture without lactate. This result suggests that KetM may utilize proton and sodium motive forces.

Intravenous peramivir inhibits viral replication, and leads to bacterial clearance and prevention of mortality during murine bacterial co-infection caused by influenza A(H1N1)pdm09 virus and Streptococcus pneumoniae.

INTRODUCTION: Influenza virus infection increases susceptibility to bacterial infection and mortality in humans. Although the efficacy of approved intravenous peramivir, a neuraminidase (NA) inhibitor, against influenza virus infection has been reported, its efficacy against bacterial co-infection, which occurs during the period of viral shedding, was not fully investigated. To further understand the significance of treatment with peramivir, we assessed the efficacy of peramivir against a bacterial co-infection model in mice caused by clinically isolated influenza A(H1N1)pdm09 virus and Streptococcus pneumoniae. METHODS: Mice were infected with influenza A(H1N1)pdm09. Peramivir was intravenously administered after the viral infection. At 2days post viral infection, the mice were infected with S. pneumoniae. Peramivir efficacy was measured by the survival rates and viral titers, bacterial titers, or proinflammatory cytokine concentrations in lung homogenates. RESULTS: Peramivir treatment reduced the mortality of mice infected with influenza virus and S. pneumoniae. The survival rate in the peramivir-treated group was significantly higher than that in the oseltamivir-treated group. Viral titers and proinflammatory cytokine responses in the peramivir-treated group were significantly lower than those in the oseltamivir-treated group until at 2days post viral infection. Bacterial titer was significantly lower in the peramivir-treated group than in the oseltamivir-treated group at 4days post viral infection. CONCLUSION: These results demonstrated that peramivir inhibits viral replication, consequently leading to bacterial clearance and prevention of mortality during severe murine bacterial co-infection, which occurs during the period of viral shedding, with the efficacy of peramivir being superior to that of oseltamivir.

Suppression of stop codon UGA in acrB can contribute to antibiotic resistance in Klebsiella pneumoniae ATCC10031.

We previously reported that Klebsiella pneumoniae MGH78578 exhibited higher resistance against various antimicrobials than K. pneumoniae ATCC10031. In this study, we showed that the plasmid, pKPN5, in K. pneumoniae MGH78578 played an important role in resistance against aminoglycosides, ampicillin, tetracycline, and chloramphenicol, while genome-derived ß-lactamases and drug efflux pumps appeared to be more important in resistance to cloxacillin. acrAB, encoding a potent multidrug efflux pump, was cloned from K. pneumoniae MGH78578 and ATCC10031, to investigate reasons for the high drug resistance of K. pneumoniae MGH78578, and the results revealed that AcrAB from K. pneumoniae ATCC10031 conferred weaker drug resistance than AcrAB from K. pneumoniae MGH78578. DNA sequencing revealed that acrB from K. pneumoniae ATCC10031 carried the nonsense mutation, UGA, which was not found in acrB from K. pneumoniae MGH78578. However, acrB from K. pneumoniae ATCC10031 conferred slightly elevated resistant levels to several antimicrobials. The intact length of AcrB was detected in K. pneumoniae ATCC10031 by Western blot analysis, even though its quantity was small. Therefore, the stop codon UGA in acrB was thought to be overcome to some extent in this strain. We artificially introduced the nonsense mutation,UGA to the cat gene on pACYC184, and the plasmid also elevated the MIC of chloramphenicol in K. pneumoniae ATCC10031. These results suggest that a mechanism to overcome the nonsense mutation in acrB sustained resistance against a few ß-lactams, dyes, and cholic acid in K. pneumoniae ATCC10031.

Hemozoin is a potent adjuvant for hemagglutinin split vaccine without pyrogenicity in ferrets.

BACKGROUND: Synthetic hemozoin (sHZ, also known as ß-hematin) from monomeric heme is a particle adjuvant which activates antigen-presenting cells (APCs), such as dendritic cells and macrophages, and enhances humoral immune responses to several antigens, including ovalbumin, human serum albumin, and serine repeat antigen 36 of Plasmodium falciparum. In the present study, we evaluated the adjuvanticity and pyrogenicity of sHZ as an adjuvant for seasonal trivalent hemagglutinin split vaccine (SV) for humans using the experimental ferret model. METHOD: Ferrets were twice immunized with trivalent SV, SV with sHZ (SV/sHZ) or Fluad, composed of trivalent SV with MF59. Serum hemagglutination inhibition (HI) titers against three viral hemagglutinin (HA) antigens were measured at every week after the immunization. The pyrogenicity of SV/sHZ was examined by monitoring the body temperature of the immunized ferrets. To evaluate the protective efficacy of SV/sHZ, the immunized ferrets were challenged with influenza virus B infection, followed by measurement of viral titers in the nasal cavity and body temperature. RESULTS: sHZ enhanced HI titers against three viral HA antigens in a dose-dependent manner, to an extent comparable to that of Fluad. The highest dose of sHZ (800 µg) immunized with SV conferred sterile protection against infection with heterologous Influenza B virus, without causing any pyrogenic reaction such as high fever. CONCLUSION: In the present study, sHZ enhanced the protective efficacy of SV against influenza infection without inducing pyrogenic reaction, suggesting sHZ to be a promising adjuvant candidate for human SV.

Functional study of the novel multidrug efflux pump KexD from Klebsiella pneumoniae.

We cloned a gene, kexD, that provides a multidrug-resistant phenotype from multidrug-resistant Klebsiella pneumoniae MGH78578. The deduced amino acid sequence of KexD is similar to that of the inner membrane protein, RND-type multidrug efflux pump. Introduction of the kexD gene into Escherichia coli KAM32 resulted in a MIC that was higher for erythromycin, novobiocin, rhodamine 6G, tetraphenylphosphonium chloride, and ethidium bromide than that of the control. Intracellular ethidium bromide levels in E. coli cells carrying the kexD gene were lower than that in the control cells under energized conditions, suggesting that KexD is a component of an energy-dependent efflux pump. RND-type pumps typically consist of three components: an inner membrane protein, a periplasmic protein, and an outer membrane protein. We discovered that KexD functions with a periplasmic protein, AcrA, from E. coli and K. pneumoniae, but not with the periplasmic proteins KexA and KexG from K. pneumoniae. KexD was able to utilize either TolC of E. coli or KocC of K. pneumoniae as an outer membrane component. kexD mRNA was not detected in K. pneumoniae MGH78578 or ATCC10031. We isolated erythromycin-resistant mutants from K. pneumoniae ATCC10031, and some showed a multidrug-resistant phenotype similar to the drug resistance pattern of KexD. Two strains of multidrug-resistant mutants were investigated for kexD expression; kexD mRNA levels were increased in these strains. We conclude that changing kexD expression can contribute to the occurrence of multidrug-resistant K. pneumoniae.

Properties and expression of a multidrug efflux pump AcrAB-KocC from Klebsiella pneumoniae.

We previously reported that we had cloned genes responsible for multidrug resistance from the chromosomal DNA of Klebsiella pneumoniae MGH78578 using a drug-hypersusceptible Escherichia coli strain as a host. One of the recombinant plasmids pETV6 conferred resistance to host cells against a wide range of antimicrobial agents, dyes and detergents. It was revealed that this plasmid carried the acrBKp gene and a part of the acrAKp gene coding for a multidrug efflux pump belonging to the RND family. We cloned the whole acrAKpBKp operon of K. pneumoniae and characterized the pump. The AcrAB pump utilized TolC as an outer membrane component in cells of E. coli. Elevated energy-dependent efflux of ethidium was observed with cells possessing AcrAKp BKp-TolC. We cloned a gene coding for an ortholog of TolC from chromosomal DNA of K. pneumoniae, and designated it kocC. It seems that the AcrAKpBKp-KocC complex functions as a potent multidrug efflux pump in K. pneumoniae. We observed a higher level of expression of acrAKp in K. pneumoniae MGH78578, a multidrug resistant strain, compared with ATCC10031, a drug susceptible strain.