Radiosynthesis automation, non-human primate biodistribution and dosimetry of K+ channel tracer [11C]3MeO4AP.

BACKGROUND: 4-Aminopyridine (4AP) is a medication for the symptomatic treatment of multiple sclerosis. Several 4AP-based PET tracers have been developed for imaging demyelination. In preclinical studies, [11C]3MeO4AP has shown promise due to its high brain permeability, high metabolic stability, high plasma availability, and high in vivo binding affinity. To prepare for the translation to human studies, we developed a cGMP-compatible automated radiosynthesis protocol and evaluated the whole-body biodistribution and radiation dosimetry of [11C]3MeO4AP in non-human primates (NHPs). METHODS: Automated radiosynthesis was carried out using a GE TRACERlab FX-C Pro synthesis module. One male and one female adult rhesus macaques were used in the study. A high-resolution CT from cranial vertex to knee was acquired. PET data were collected using a dynamic acquisition protocol with four bed positions and 13 passes over a total scan time of ~ 150 min. Based on the CT and PET images, volumes of interest (VOIs) were manually drawn for selected organs. Non-decay corrected time-activity curves (TACs) were extracted for each VOI. Radiation dosimetry and effective dose were calculated from the integrated TACs using OLINDA software. RESULTS: Fully automated radiosynthesis of [11C]3MeO4AP was achieved with 7.3 ± 1.2% (n = 4) of non-decay corrected radiochemical yield within 38 min of synthesis and purification time. [11C]3MeO4AP distributed quickly throughout the body and into the brain. The organs with highest dose were the kidneys. The average effective dose of [11C]3MeO4AP was 4.0 ± 0.6 µSv/MBq. No significant changes in vital signs were observed during the scan. CONCLUSION: A cGMP-compatible automated radiosynthesis of [11C]3MeO4AP was developed. The whole-body biodistribution and radiation dosimetry of [11C]3MeO4AP was successfully evaluated in NHPs. [11C]3MeO4AP shows lower average effective dose than [18F]3F4AP and similar average effective dose as other carbon-11 tracers.

PET imaging of M4 muscarinic acetylcholine receptors in rhesus macaques using [11C]MK-6884: Quantification with kinetic modeling and receptor occupancy by CVL-231 (emraclidine), a novel positive allosteric modulator.

Stimulation of the M4 muscarinic acetylcholine receptor reduces striatal hyperdopaminergia, suggesting its potential as a therapeutic target for schizophrenia. Emraclidine (CVL-231) is a novel, highly selective, positive allosteric modulator (PAM) of M4 muscarinic acetylcholine receptors i.e. acts as a modulator that increases the response of these receptors. First, we aimed to further characterize the positron emission tomography (PET) imaging and quantification performance of a recently developed M4 PAM radiotracer, [11C]MK-6884, in non-human primates (NHPs). Second, we applied these results to determine the receptor occupancy of CVL-231 as a function of dose. Using paired baseline-blocking PET scans, we quantified total volume of distribution, binding potential, and receptor occupancy. Both blood-based and reference region-based methods quantified M4 receptor levels across brain regions. The 2-tissue 4-parameter kinetic model best fitted regional [11C]MK-6884-time activity curves. Only the caudate nucleus and putamen displayed statistically significant [11C]MK-6884 uptake and dose-dependent blocking by CVL-231. For binding potential and receptor occupancy quantification, the simplified reference tissue model using the grey cerebellum as a reference region was employed. CVL-231 demonstrated dose-dependent M4 receptor occupancy in the striatum of the NHP brain and shows promise for further development in clinical trials.

Preclinical efficacy of a cell division protein candidate gonococcal vaccine identified by artificial intelligence.

A safe and effective vaccine is urgently needed to combat the global threat of multidrug-resistant (MDR) Neisseria gonorrhoeae. We screened 26 gonococcal proteins discovered by an artificial intelligence-driven platform called Efficacy Discriminative Educated Network (EDEN) trained to identify novel, protective vaccine antigens against pathogenic bacteria for efficacy in the mouse vaginal colonization model of gonorrhea. Combinations of two to three antigens adjuvanted with GLA-SE (induces TH1 responses) yielded 11 groups that were used to vaccinate mice. An inverse correlation was noted between the complement-dependent bactericidal activity of antisera from each of the 11 groups and the burden of gonococcal colonization. The combination of NGO1549 (FtsN; cell divisome protein) and NGO0265 (predicted cell division protein) most substantially reduced the burden of colonization by MDR strain WHO X. The EDEN prediction score for each group of antigens correlated positively with reductions in overall bacterial burden, providing evidence for its predictive potential. FtsN and NGO0265 administered either individually, in combination, or as a chimeric protein significantly attenuated gonococcal vaginal colonization by all three test strains. IgG in antisera from mice immunized with the chimeric NGO0265-FtsN protein supported the complement-dependent killing of all 50 (100%) gonococcal isolates tested. The efficacy of the chimeric NGO0265-FtsN vaccine required the membrane attack complex (C5b-9) of complement, evidenced by loss of efficacy in complement C9-/- mice. In conclusion, a chimeric molecule comprising NGO0265 and FtsN adjuvanted with GLA-SE elicits IgG with broad anti-gonococcal bactericidal activity, attenuates gonococcal colonization in a complement-dependent manner, and represents a promising gonococcal vaccine candidate.IMPORTANCEVaccines to curb the global spread of multidrug-resistant gonorrhea are urgently needed. Here, 26 vaccine candidates identified by an artificial intelligence-driven platform (Efficacy Discriminative Educated Network[EDEN]) were screened for efficacy in the mouse vaginal colonization model. Complement-dependent bactericidal activity of antisera and the EDEN protective scores both correlated positively with the reduction in overall bacterial colonization burden. NGO1549 (FtsN) and NGO0265, both involved in cell division, displayed the best activity and were selected for further development. Both antigens, when fused to create a chimeric protein, elicited bactericidal antibodies against a wide array of gonococcal isolates and significantly attenuated the duration and burden of gonococcal colonization of mouse vaginas. Protection was abrogated in mice that lacked complement C9, the last step in the formation of the membrane attack complex pore, suggesting complement-dependent bactericidal activity as a mechanistic correlate of protection of the vaccine. FtsN and NGO0265 represent promising vaccine candidates against gonorrhea.

Increase in Multidrug Resistant Neisseria gonorrhoeae FC428-Like Isolates Harboring the Mosaic penA 60.001 Gene, in Nanjing, China (2017-2020).

Background: Since the first Chinese report of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone in 2016, additional FC428-like, penA 60.001 isolates have been identified in China. Objective: To document the rise in penA 60.001 isolates in Nanjing, China, and characterize their molecular and epidemiological features. Methods: N. gonorrhoeae minimum inhibitory concentrations (MICs, mg/L) for ceftriaxone, cefixime, penicillin, tetracycline, ciprofloxacin, azithromycin, spectinomycin, gentamicin and zoliflodacin were determined by agar dilution. MICs for ertapenem were measured by E-test. N. gonorrhoeae antimicrobial sequence typing (NG-STAR) of seven loci (penA, mtrR, porB, ponA, gyrA, parC and 23S rRNA) was analyzed together with N. gonorrhoeae multiantigen sequence typing (NG-MAST) and multilocus sequence typing (MLST). Phylogenetic analysis was also performed using whole genomic sequencing (WGS). Results: Fourteen FC428-related penA 60.001 N. gonorrhoeae infections were identified out of 677 infections from 2017 to 2020, in Nanjing, representing an incremental yearly rise in the percentage of the city's N. gonorrhoeae isolates that were FC428-related. Seven FC428-related N. gonorrhoeae infections were acquired in Nanjing, proper; four others in eastern Chinese cities and three from unknown locations. All FC428-related isolates were resistant to ceftriaxone, cefixime, ciprofloxacin, tetracycline and penicillin but susceptible to spectinomycin, gentamicin, ertapenem and zoliflodacin; three strains were resistant to azithromycin. penA 60.001 isolates displayed closely related MLST types and NG-STAR types but relatively distant NG-MAST types. WGS showed a phylogenetic analysis that intermingled with other international isolates. Conclusion: penA 60.001 N. gonorrhoeae isolates emerged in Nanjing, China, beginning in 2017, and have continued to rise.

Human biodistribution and radiation dosimetry of the demyelination tracer [18F]3F4AP.

PURPOSE: [18F]3F4AP is a novel PET radiotracer that targets voltage-gated potassium (K+) channels and has shown promise for imaging demyelinated lesions in animal models of neurological diseases. This study aimed to evaluate the biodistribution, safety, and radiation dosimetry of [18F]3F4AP in healthy human volunteers. METHODS: Four healthy volunteers (2 females) underwent a 4-h dynamic PET scan from the cranial vertex to mid-thigh using multiple bed positions after administration of 368 ± 17.9 MBq (9.94 ± 0.48 mCi) of [18F]3F4AP. Volumes of interest for relevant organs were manually drawn guided by the CT, and PET images and time-activity curves (TACs) were extracted. Radiation dosimetry was estimated from the integrated TACs using OLINDA software. Safety assessments included measuring vital signs immediately before and after the scan, monitoring for adverse events, and obtaining a comprehensive metabolic panel and electrocardiogram within 30 days before and after the scan. RESULTS: [18F]3F4AP distributed throughout the body with the highest levels of activity in the kidneys, urinary bladder, stomach, liver, spleen, and brain and with low accumulation in muscle and fat. The tracer cleared quickly from circulation and from most organs. The clearance of the tracer was noticeably faster than previously reported in nonhuman primates (NHPs). The average effective dose (ED) across all subjects was 12.1 ± 2.2 µSv/MBq, which is lower than the estimated ED from the NHP studies (21.6 ± 0.6 µSv/MBq) as well as the ED of other fluorine-18 radiotracers such as [18F]FDG (~ 20 µSv/MBq). No differences in ED between males and females were observed. No substantial changes in safety assessments or adverse events were recorded. CONCLUSION: The biodistribution and radiation dosimetry of [18F]3F4AP in humans are reported for the first time. The average total ED across four subjects was lower than most 18F-labeled PET tracers. The tracer and study procedures were well tolerated, and no adverse events occurred.

Neisseria gonorrhoeae Infection in Women Increases With Rising Gonococcal Burdens in Partners: Chlamydia Coinfection in Women Increases Gonococcal Burden.

BACKGROUND: Likelihood of Neisseria gonorrhoeae infection in women exposed to male sex partners with increasing N. gonorrhoeae burdens and enhancement by Chlamydia trachomatis is not defined. METHODS: We identified men with urethritis and their regular female sex partners. Exposure to N. gonorrhoeae burdens in men was compared in N. gonorrhoeae-infected versus -uninfected partners. Association of N. gonorrhoeae infection in women with burdens in male partners was estimated using logistic regression. Association of C. trachomatis coinfection and N. gonorrhoeae burdens in women adjusted for burdens in male partners was estimated by linear regression. RESULTS: In total, 1816 men were enrolled; 202 had ≥2 partners, 91 who confirmed monogamy and were enrolled; 77% were married. Seventy were partners of N. gonorrhoeae-infected men; 58 (83%) were N. gonorrhoeae infected, 26 (45%) C. trachomatis coinfected. Infected women had partners with 9.3-fold higher N. gonorrhoeae burdens than partners of uninfected women (P = .0041). Association of N. gonorrhoeae infection in women with upper quartiles of N. gonorrhoeae burdens in partners increased (odds ratios ≥ 2.97)compared to the first quartile (P = .032). N. gonorrhoeae burdens in C. trachomatis-coinfected women were 2.82-fold higher than in C. trachomatis-uninfected women (P = .036). CONCLUSIONS: N. gonorrhoeae infections increased in women whose partners were infected with higher N. gonorrhoeae burdens. C. trachomatis coinfection was associated with increased N. gonorrhoeae burdens in women.

In Vitro Activity of Ertapenem against Neisseria gonorrhoeae Clinical Isolates with Decreased Susceptibility or Resistance to Extended-Spectrum Cephalosporins in Nanjing, China (2013 to 2019).

Neisseria gonorrhoeae isolates collected in Nanjing, China, that possessed decreased susceptibility (or resistance) to extended-spectrum cephalosporins (ESCs) were examined for susceptibility to ertapenem, and their sequence types were determined. Ceftriaxone and cefixime MICs of ≥0.125 mg/L and ≥0.25 mg/L, respectively, were first determined in 259 strains isolated between 2013 and 2019, and then MICs of ertapenem were measured using the antimicrobial gradient Epsilometer test (Etest). Also, genetic determinants of ESC resistance were identified and N. gonorrhoeae multiantigen sequence typing (NG-MAST) was performed to analyze associations with ertapenem susceptibility. All isolates displayed ertapenem MICs between 0.006 mg/L and 0.38 mg/L; the overall MIC50 and MIC90 were 0.032 mg/L and 0.125 mg/L, respectively. Forty-four (17.0%) isolates displayed ertapenem MICs of ≥0.125 mg/L; 10 (3.9%) had MICs of ≥0.25 mg/L. The proportion of isolates with ertapenem MICs of ≥0.125 mg/L increased from 4.0% in 2013 to 20.0% in 2019 (χ2 = 24.144, P < 0.001; chi-square test for linear trend). The penA mosaic allele was present in a significantly higher proportion of isolates with ertapenem MICs of ≥0.125 mg/L than of isolates with MICs of ≤0.094 mg/L) (97.7% versus 34.9%, respectively; χ2 = 58.158, P < 0.001). ST5308 was the most prevalent NG-MAST type (8.5%); ST5308 was also significantly more common among isolates with ertapenem MICs of ≥0.125 mg/L than isolates with MICs of ≤0.094 mg/L (22.7% and 5.6%, respectively; χ2 = 13.815, P = 0.001). Ertapenem may be effective therapy for gonococcal isolates with decreased susceptibility or resistance to ESCs and isolates with identifiable genetic resistance determinants.

Efficacy of an Experimental Gonococcal Lipooligosaccharide Mimitope Vaccine Requires Terminal Complement.

A safe and effective vaccine against multidrug-resistant gonorrhea is urgently needed. An experimental peptide vaccine called TMCP2 that mimics an oligosaccharide epitope in gonococcal lipooligosaccharide, when adjuvanted with glucopyranosyl lipid adjuvant-stable emulsion, elicits bactericidal immunoglobulin G and hastens clearance of gonococci in the mouse vaginal colonization model. In this study, we show that efficacy of TMCP2 requires an intact terminal complement pathway, evidenced by loss of activity in C9-/- mice or when C7 function was blocked. In conclusion, TMCP2 vaccine efficacy in the mouse vagina requires membrane attack complex. Serum bactericidal activity may serve as a correlate of protection for TMCP2.

In Vitro Efficacy of Gentamicin Alone and in Combination with Ceftriaxone, Ertapenem, and Azithromycin against Multidrug-Resistant Neisseria gonorrhoeae.

This study was conducted to determine the in vitro activities of gentamicin alone and in combination with ceftriaxone, ertapenem, and azithromycin against multidrug-resistant (MDR) Neisseria gonorrhoeae isolates. A total of 407 clinical isolates from Nanjing, China, obtained in 2016 to 2017, had MICs determined for gentamicin using the agar dilution method. MDR status was ascribed to 97 strains that displayed decreased susceptibility or resistance to extended-spectrum cephalosporins (ESCs) (ceftriaxone [MIC, ≥0.125 mg/liter] and cefixime [MIC, ≥0.25 mg/liter]), plus resistance to at least two of the following antimicrobials: penicillin (MIC, ≥2 mg/liter), ciprofloxacin (MIC, ≥1 mg/liter), and azithromycin (MIC, ≥1 mg/liter). MDR strains underwent MIC determinations for antimicrobial combinations using the antimicrobial gradient epsilometer test (Etest). Results that ranged from synergy to antagonism were interpreted using the fractional inhibitory concentration (FICI). All 407 gonococcal isolates were susceptible to gentamicin; MICs ranged from 2 mg/liter to 16 mg/liter. Synergy was demonstrated in 16.5% (16/97), 27.8% (27/97), and 8.2% (8/97) of MDR strains when gentamicin was combined with ceftriaxone (geometric mean [GM] FICI, 0.747), ertapenem (GM FICI, 0.662), and azithromycin (GM FICI, 1.021), respectively. No antimicrobial antagonism was observed with any combination tested against MDR strains; overall, antimicrobial combinations were indifferent. The GM MICs of gentamicin were reduced by 2.63-, 3.80-, and 1.98-fold when tested in combination with ceftriaxone, ertapenem, and azithromycin, respectively. The GM MICs of the three additional antimicrobials individually were reduced by 3-, 2.57-, and 1.98-fold, respectively, when each was tested in combination with gentamicin. Gentamicin alone was effective in vitro against N. gonorrhoeae, including MDR isolates. Combination testing of MDR strains showed lower MICs against gentamicin and each of three antimicrobials (ceftriaxone, ertapenem, and azithromycin) when used in combination. IMPORTANCE Antimicrobial-resistant Neisseria gonorrhoeae is a major global public health concern. New treatment options are urgently needed to successfully treat multidrug-resistant (MDR) Neisseria gonorrhoeae infections. This study showed that gentamicin maintained excellent in vitro susceptibility against clinical gonococcal isolates collected in 2016 and 2017, including MDR isolates. Combinations of gentamicin plus ertapenem, ceftriaxone, and azithromycin produced synergistic effects against certain MDR isolates. No antagonism was observed in any of the antimicrobial combinations, which may prove useful to guide clinical testing of combination therapies.

Synthetic DNA Delivery of an Optimized and Engineered Monoclonal Antibody Provides Rapid and Prolonged Protection against Experimental Gonococcal Infection.

Monoclonal antibody (MAb) 2C7 recognizes a lipooligosaccharide epitope expressed by most clinical Neisseria gonorrhoeae isolates and mediates complement-dependent bactericidal activity. We recently showed that a recombinant human IgG1 chimeric variant of MAb 2C7 containing an E430G Fc modification (2C7_E430G), which enhances complement activation, outperformed the parental MAb 2C7 (2C7_WT) in vivo Because natural infection with N. gonorrhoeae often does not elicit protective immunity and reinfections are common, approaches that prolong bacterial control in vivo are of great interest. Advances in DNA-based approaches have demonstrated the combined benefit of genetic engineering, formulation optimizations, and facilitated delivery via CELLECTRA-EP technology, which can induce robust in vivo expression of protective DNA-encoded monoclonal antibodies (DMAbs) with durable serum activity relative to traditional recombinant MAb therapies. Here, we created optimized 2C7-derived DMAbs encoding the parental Fc (2C7_WT) or complement-enhancing Fc variants (2C7_E430G and 2C7_E345K). 2C7 DMAbs were rapidly generated and detected throughout the 4-month study. While all complement-engaging 2C7 variants facilitated rapid clearance following primary N. gonorrhoeae challenge (day 8 after DMAb administration), the complement-enhancing 2C7_E430G variant demonstrated significantly higher potency against mice rechallenged 65 days after DMAb administration. Passive intravenous transfer of in vivo-produced, purified 2C7 DMAbs confirmed the increased potency of the complement-enhancing variants. This study highlights the ability of the DMAb platform to launch the in vivo production of antibodies engineered to promote and optimize downstream innate effector mechanisms such as complement-mediated killing, leading to hastened bacterial elimination.IMPORTANCENeisseria gonorrhoeae has become resistant to most antibiotics in clinical use. Currently, there is no safe and effective vaccine against gonorrhea. Measures to prevent the spread of gonorrhea are a global health priority. A monoclonal antibody (MAb) called 2C7, directed against a lipooligosaccharide glycan epitope expressed by most clinical isolates, displays complement-dependent bactericidal activity and hastens clearance of gonococcal vaginal colonization in mice. Fc mutations in a human IgG1 chimeric version of MAb 2C7 further enhance complement activation, and the resulting MAb displays greater activity than wild-type MAb 2C7 in vivo Here, we utilized a DNA-encoded MAb (DMAb) construct designed to launch production and assembly of "complement-enhanced" chimeric MAb 2C7 in vivo The ensuing rapid and sustained MAb 2C7 expression attenuated gonococcal colonization in mice at 8 days as well as 65 days postadministration. The DMAb system may provide an effective, economical platform to deliver MAbs for durable protection against gonorrhea.

Susceptibility Trends of Zoliflodacin against Multidrug-Resistant Neisseria gonorrhoeae Clinical Isolates in Nanjing, China, 2014 to 2018.

Previously, we reported the potent activity of a novel spiropyrimidinetrione, zoliflodacin, against Neisseria gonorrhoeae isolates collected in 2013 from symptomatic men in Nanjing, China. Here, we investigated trends of susceptibilities to zoliflodacin in 986 isolates collected from men between 2014 and 2018. N. gonorrhoeae isolates were tested for susceptibility to zoliflodacin and seven other antibiotics. Mutations in the gyrA, gyrB, parC, parE, and mtrR genes were determined by PCR and sequencing. The MICs of zoliflodacin ranged from ≤0.002 to 0.25 mg/liter; the overall MIC50 and MIC90 were 0.06 mg/liter and 0.125 mg/liter, respectively, in 2018, increasing 2-fold from 2014. However, the percentage of isolates with lower zoliflodacin MICs declined in each year sequentially, while the percentage with higher MICs increased yearly (P ≤ 0.00001). All isolates were susceptible to spectinomycin but resistant to ciprofloxacin (MIC ≥ 1 mg/liter); 21.2% (209/986) were resistant to azithromycin (≥1 mg/liter), 43.4% (428/986) were penicillinase-producing N. gonorrhoeae (PPNG), 26.9% (265/986) were tetracycline-resistant N. gonorrhoeae (TRNG), and 19.4% (191/986) were multidrug-resistant (MDR) isolates. 202 isolates with the lowest (≤0.002 to 0.015 mg/liter) and highest (0.125 to 0.25 mg/liter) zoliflodacin MICs were quinolone resistant with double or triple mutations in gyrA; 193/202 (95.5%) also had mutations in parC There were no D429N/A and/or K450T mutations in GyrB identified in the 143 isolates with higher zoliflodacin MICs; an S467N mutation in GyrB was identified in one isolate. We report that zoliflodacin continues to have excellent in vitro activity against clinical gonococcal isolates, including those with high-level resistance to ciprofloxacin, azithromycin, and extended-spectrum cephalosporins.

Efficacy of Antigonococcal CMP-Nonulosonate Therapeutics Require Cathelicidins.

Novel therapies to counteract multidrug-resistant gonorrhea are urgently needed. A unique gonococcal immune evasion strategy involves capping of lipooligosaccharide (LOS) with sialic acid by gonococcal sialyltransferase (Lst), utilizing host-derived CMP-sialic acid (CMP-Neu5Ac in humans). LOS sialylation renders gonococci resistant to complement and cationic peptides, and down-regulates the inflammatory response by engaging siglecs. CMP-sialic acid analogs (CMP-nonulosonates [CMP-NulOs]) such as CMP-Leg5,7Ac2 and CMP-Kdn are also utilized by Lst. Incorporation of these NulO analogs into LOS maintains gonococci susceptible to complement. Intravaginal administration of CMP-Kdn or CMP-Leg5,7Ac2 attenuates gonococcal colonization of mouse vaginas. Here, we identify a key mechanism of action for the efficacy of CMP-NulOs. Surprisingly, CMP-NulOs remained effective in complement C1q-/- and C3-/- mice. LOS Neu5Ac, but not Leg5,7Ac2 or Kdn, conferred resistance to the cathelicidins LL-37 (human) and mouse cathelicidin-related antimicrobial peptide in vitro. CMP-NulOs were ineffective in Camp-/- mice, revealing that cathelicidins largely mediate the efficacy of therapeutic CMP-NulOs.

Development, validation and regulatory acceptance of improved purification and simplified quality control of [13N] Ammonia.

BACKGROUND: [13N]Ammonia is a cyclotron produced myocardial perfusion imaging agent. With the development of high-yielding [13N]ammonia cyclotron targets using a solution of 5 mM ethanol in water, there was a need to develop and validate an automated purification and formulation system for [13N]ammonia to be in a physiological compatible formulation of 0.9% sodium chloride since there is no widely available commercial system at this time. Due to its short half-life of 10 min, FDA and USP regulations allow [13N]ammonia to be tested in quality control (QC) sub-batches with limited quality control testing performed on the sub-batches for patient use. The current EP and the original USP method for the determination of the radiochemical purity and identity of [13N]ammonia depended on an HPLC method using a conductivity detector and a solvent free of other salts. This HPLC method created issues in a modern cGMP high volume PET manufacturing facility where the HPLC is used with salt containing mobile phase buffers for quality control analysis of other PET radiopharmaceuticals. Flushing of the HPLC system of residual salt buffers which may interfere with the [13N]ammonia assay can take several hours of instrument time. Since there are no mass limits on [13N]ammonia, a simplified TLC assay to determine radiochemical identity and purity could be developed to simplify and streamline QC. RESULTS: We have developed and validated a streamlined automated synthesis for [13N]ammonia which provides the drug product in 8 mL of 0.9% sodium chloride for injection. A novel radio-TLC method was developed and validated to demonstrate feasibility to quantitate [13N]ammonia and separate it from all known radiochemical impurities. CONCLUSIONS: The process for automated synthesis of [13N]ammonia simplifies and automates the purification and formulation of [13N]ammonia in a cGMP compliant manner needed for high-throughput manufacture of [13N]ammonia. The novel radio-TLC method has simplified [13N]ammonia quality control (QC) and now enables it to be tested using the same QC equipment as [18F]fludeoxyglucose (FDA/USP recognized name for 2-[18F]fluoro-2-deoxy-D-glucose). Both the streamlined automated synthesis of [13N]ammonia and the novel radio-TLC method have been accepted and approved by the US Food and Drug Administration (FDA) for the cGMP manufacture of [13N]ammonia.

Mycoplasma genitalium in Symptomatic Male Urethritis: Macrolide Use Is Associated With Increased Resistance.

BACKGROUND: Mycoplasma genitalium (MG) causes symptomatic urethritis in men, and can infect alone or together with other sexually transmitted infection (STI) agents. METHODS: The prevalence of MG and other STIs was determined in 1816 men with symptomatic urethritis. Resistance of MG to macrolides and fluoroquinolones was determined by sequencing; the impact of recent antimicrobial usage on the distribution of MG single or mixed infections was determined. RESULTS: Overall, prevalence of MG infection was 19.7% (358/1816). Fifty-four percent (166/307) of MG infections occurred alone in the absence of other STI agents. Men with single MG infection self-administered or were prescribed antibiotics more often in the 30 days prior to enrollment than subjects with urethritis caused by MG coinfection (P < .0001). Higher rates (96.7%) of infection with macrolide resistance in MG were identified in men who had taken macrolides prior to enrollment (P < .03). Overall, 88.9% (303/341) of 23S ribosomal RNA (rRNA) genes contained mutations responsible for macrolide resistance; 89.5% (308/344) of parC and 12.4% (42/339) of gyrA genes had mutations responsible for fluoroquinolone resistance. Approximately 88% (270/308) of MG had combined mutations in 23S rRNA and parC genes; 10.4% (32/308) had mutations in all 3 genes. CONCLUSIONS: MG was the single pathogen identified in 11% of men with symptomatic urethritis. Overall, nearly 90% of MG infections were resistant to macrolides and fluoroquinolones. Men who took macrolides in the 30 days prior to enrollment had higher rates (97%) of macrolide-resistant MG. Resistance was associated with numerous mutations in 23SrRNA, parC, and gyrA genes.

Bypassing Phase Variation of Lipooligosaccharide (LOS): Using Heptose 1 Glycan Mutants To Establish Widespread Efficacy of Gonococcal Anti-LOS Monoclonal Antibody 2C7.

The sialylatable lacto-N-neotetraose (LNnT; Gal-GlcNAc-Gal-Glc) moiety from heptose I (HepI) of the lipooligosaccharide (LOS) of Neisseria gonorrhoeae undergoes positive selection during human infection. Lactose (Gal-Glc) from HepII, although phase variable, is commonly expressed in humans; loss of HepII lactose compromises gonococcal fitness in mice. Anti-LOS monoclonal antibody (MAb) 2C7, a promising antigonococcal immunotherapeutic that elicits complement-dependent bactericidal activity and attenuates gonococcal colonization in mice, recognizes an epitope comprised of lactoses expressed simultaneously from HepI and HepII. Glycan extensions beyond lactose on HepI modulate binding and function of MAb 2C7 in vitro Here, four gonococcal LOS mutants, each with lactose from HepII but fixed (unable to phase-vary) LOS HepI glycans extended beyond the lactose substitution of HepI (lactose alone, Gal-lactose, LNnT, or GalNAc-LNnT), were used to define how HepI glycan extensions affect (i) mouse vaginal colonization and (ii) efficacy in vitro and in vivo of a human IgG1 chimeric derivative of MAb 2C7 (2C7-Ximab) with a complement-enhancing E-to-G Fc mutation at position 430 (2C7-Ximab-E430G). About 10-fold lower 2C7-Ximab-E430G concentrations achieved similar complement-dependent killing of three gonococcal mutants with glycan extensions beyond lactose-substituted HepI (lactose alone, LNnT, or GalNAc-LNnT) as 2C7-Ximab (unmodified Fc). The fourth mutant (Gal-lactose) resisted direct complement-dependent killing but was killed approximately 70% by 2C7-Ximab-E430G in the presence of polymorphonuclear leukocytes and complement. Only mutants with (sialylatable) LNnT from HepI colonized mice for >3 days, reiterating the importance of LNnT sialylation for infection. 2C7-Ximab-E430G significantly attenuated colonization caused by the virulent mutants.

Preclinical Efficacy of a Lipooligosaccharide Peptide Mimic Candidate Gonococcal Vaccine.

The global spread of multidrug-resistant strains of Neisseria gonorrhoeae constitutes a public health emergency. With limited antibiotic treatment options, there is an urgent need for development of a safe and effective vaccine against gonorrhea. Previously, we constructed a prototype vaccine candidate comprising a peptide mimic (mimitope) of a glycan epitope on gonococcal lipooligosaccharide (LOS), recognized by monoclonal antibody 2C7. The 2C7 epitope is (i) broadly expressed as a gonococcal antigenic target in human infection, (ii) a critical requirement for gonococcal colonization in the experimental setting, and (iii) a virulence determinant that is maintained and expressed by gonococci. Here, we have synthesized to >95% purity through a relatively facile and economical process a tetrapeptide derivative of the mimitope that was cyclized through a nonreducible thioether bond, thereby rendering the compound homogeneous and stable. This vaccine candidate, called TMCP2, when administered at 0, 3, and 6 weeks to BALB/c mice at either 50, 100 or 200 µg/dose in combination with glucopyranosyl lipid A-stable oil-in-water nanoemulsion (GLA-SE; a Toll-like receptor 4 and TH1-promoting adjuvant), elicited bactericidal IgG and reduced colonization levels of gonococci in experimentally infected mice while accelerating clearance by each of two different gonococcal strains. Similarly, a 3-dose biweekly schedule (50 µg TMCP2/dose) was also effective in mice. We have developed a gonococcal vaccine candidate that can be scaled up and produced economically to a high degree of purity. The candidate elicits bactericidal antibodies and is efficacious in a preclinical experimental infection model.IMPORTANCENeisseria gonorrhoeae has become resistant to most antibiotics. The incidence of gonorrhea is also sharply increasing. A safe and effective antigonococcal vaccine is urgently needed. Lipooligosaccharide (LOS), the most abundant outer membrane molecule, is indispensable for gonococcal pathogenesis. A glycan epitope on LOS that is recognized by monoclonal antibody (MAb) 2C7 (called the 2C7 epitope) is expressed almost universally by gonococci in vivo Previously, we identified a peptide mimic (mimitope) of the 2C7 epitope, which when configured as an octamer and used as an immunogen, attenuated colonization of mice by gonococci. Here, a homogenous, stable tetrameric derivative of the mimitope, when combined with a TH1-promoting adjuvant and used as an immunogen, also effectively attenuates gonococcal colonization of mice. This candidate peptide vaccine can be produced economically, an important consideration for gonorrhea, which affects socioeconomically underprivileged populations disproportionately, and represents an important advance in the development of a gonorrhea vaccine.

Complement alone drives efficacy of a chimeric antigonococcal monoclonal antibody.

Multidrug-resistant Neisseria gonorrhoeae is a global health problem. Monoclonal antibody (mAb) 2C7 recognizes a gonococcal lipooligosaccharide epitope that is expressed by >95% of clinical isolates and hastens gonococcal vaginal clearance in mice. Chimeric mAb 2C7 (human immunoglobulin G1 [IgG1]) with an E430G Fc modification that enhances Fc:Fc interactions and hexamerization following surface-target binding and increases complement activation (HexaBody technology) showed significantly greater C1q engagement and C4 and C3 deposition compared to mAb 2C7 with wild-type Fc. Greater complement activation by 2C7-E430G Fc translated to increased bactericidal activity in vitro and, consequently, enhanced efficacy in mice, compared with "Fc-unmodified" chimeric 2C7. Gonococci bind the complement inhibitors factor H (FH) and C4b-binding protein (C4BP) in a human-specific manner, which dampens antibody (Ab)-mediated complement-dependent killing. The variant 2C7-E430G Fc overcame the barrier posed by these inhibitors in human FH/C4BP transgenic mice, for which a single 1 µg intravenous dose cleared established infection. Chlamydia frequently coexists with and exacerbates gonorrhea; 2C7-E430G Fc also proved effective against gonorrhea in gonorrhea/chlamydia-coinfected mice. Complement activation alone was necessary and sufficient for 2C7 function, evidenced by the fact that (1) "complement-inactive" Fc modifications that engaged Fc gamma receptor (FcγR) rendered 2C7 ineffective, nonetheless; (2) 2C7 was nonfunctional in C1q-/- mice, when C5 function was blocked, or in C9-/- mice; and (3) 2C7 remained effective in neutrophil-depleted mice and in mice treated with PMX205, a C5a receptor (C5aR1) inhibitor. We highlight the importance of complement activation for antigonococcal Ab function in the genital tract. Elucidating the correlates of protection against gonorrhea will inform the development of Ab-based gonococcal vaccines and immunotherapeutics.

Biology of the Gonococcus: Disease and Pathogenesis.

Neisseria gonorrhoeae infection is a major public health problem worldwide. The increasing incidence of gonorrhea coupled with global spread of multidrug-resistant isolates of gonococci has ushered in an era of potentially untreatable infection. Gonococcal disease elicits limited immunity, and individuals are susceptible to repeated infections. In this chapter, we describe gonococcal disease and epidemiology and the structure and function of major surface components involved in pathogenesis. We also discuss the mechanisms that gonococci use to evade host immune responses and the immune responses following immunization with selected bacterial components that may overcome evasion. Understanding the biology of the gonococcus may aid in preventing the spread of gonorrhea and also facilitate the development of gonococcal vaccines and treatments.