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

ABSTRACT 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.

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The Lst Sialyltransferase of Neisseria gonorrhoeae Can Transfer Keto-Deoxyoctanoate as the Terminal Sugar of Lipooligosaccharide: a Glyco-Achilles Heel That Provides a New Strategy for Vaccines to Prevent Gonorrhea

mBio ◽

10.1128/mbio.03666-20 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Freda E.-C. Jen ◽

Margaret R. Ketterer ◽

Evgeny A. Semchenko ◽

Christopher J. Day ◽

Kate L. Seib ◽

...

Keyword(s):

Monoclonal Antibody ◽

Neisseria Gonorrhoeae ◽

Phase Variation ◽

Multidrug Resistant ◽

Content Type ◽

Acetylneuraminic Acid ◽

New Strategy ◽

Opsonophagocytic Killing ◽

Further Development

ABSTRACT The lipooligosaccharide (LOS) of Neisseria gonorrhoeae plays key roles in pathogenesis and is composed of multiple possible glycoforms. These glycoforms are generated by the process of phase variation and by differences in the glycosyltransferase gene content of particular strains. LOS glycoforms of N. gonorrhoeae can be terminated with an N-acetylneuraminic acid (Neu5Ac), which imparts resistance to the bactericidal activity of serum. However, N. gonorrhoeae cannot synthesize the CMP-Neu5Ac required for LOS biosynthesis and must acquire it from the host. In contrast, Neisseria meningitidis can synthesize endogenous CMP-Neu5Ac, the donor molecule for Neu5Ac, which is a component of some meningococcal capsule structures. Both species have an almost identical LOS sialyltransferase, Lst, that transfers Neu5Ac from CMP-Neu5Ac to the terminus of LOS. Lst is homologous to the LsgB sialyltransferase of nontypeable Haemophilus influenzae (NTHi). Studies in NTHi have demonstrated that LsgB can transfer keto-deoxyoctanoate (KDO) from CMP-KDO to the terminus of LOS in place of Neu5Ac. Here, we show that Lst can also transfer KDO to LOS in place of Neu5Ac in both N. gonorrhoeae and N. meningitidis. Consistent with access to the pool of CMP-KDO in the cytoplasm, we present data indicating that Lst is localized in the cytoplasm. Lst has previously been reported to be localized on the outer membrane. We also demonstrate that KDO is expressed as a terminal LOS structure in vivo in samples from infected women and further show that the anti-KDO monoclonal antibody 6E4 can mediate opsonophagocytic killing of N. gonorrhoeae. Taken together, these studies indicate that KDO expressed on gonococcal LOS represents a new antigen for the development of vaccines against gonorrhea. IMPORTANCE The emergence of multidrug-resistant N. gonorrhoeae strains that are resistant to available antimicrobials is a current health emergency, and no vaccine is available to prevent gonococcal infection. Lipooligosaccharide (LOS) is one of the major virulence factors of N. gonorrhoeae. The sialic acid N-acetylneuraminic acid (Neu5Ac) is present as the terminal glycan on LOS in N. gonorrhoeae. In this study, we made an unexpected discovery that KDO can be incorporated as the terminal glycan on LOS of N. gonorrhoeae by the alpha-2,3-sialyltransferase Lst. We showed that N. gonorrhoeae express KDO on LOS in vivo and that the KDO-specific monoclonal antibody 6E4 can direct opsonophagocytic killing of N. gonorrhoeae. These data support further development of KDO-LOS structures as vaccine antigens for the prevention of infection by N. gonorrhoeae.

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Polymorphonuclear Leukocytes Prepared by Continuous-Flow Filtration Leukapheresis: Viability and Function

Blood ◽

10.1182/blood.v44.5.707.707 ◽

1974 ◽

Vol 44 (5) ◽

pp. 707-713 ◽

Cited By ~ 23

Author(s):

Michael B. Harris ◽

Isaac Djerassi ◽

Elias Schwartz ◽

Richard K. Root

Keyword(s):

Continuous Flow ◽

Polymorphonuclear Leukocytes ◽

Cell Types ◽

High Yield ◽

Trypan Blue Exclusion ◽

Flow Filtration ◽

And Function ◽

Combating Infection

Abstract Preparation of granulocytes for transfusion in high yield and relatively free of contamination by other cell types has been made possible by the technique of continuous-flow filtration leukapheresis (CFFL). Since previous work suggested that granulocytes collected in this manner may have impaired viability and function, a detailed study of the bactericidal, metabolic, and chemotactic properties of such cells was performed and compared to control cells obtained from the same donors prior to CFFL. The granulocyte percentage of the cell suspensions obtained by CFFL averaged 94.5% ± 1.5% compared to 82.5% ± 1.8% for the controls (p < 0.001) with viability of the PMNs determined by trypan blue exclusion being 97.5% ± 0.9% and 98.2% ± 0.5%, respectively. The phogocytic, metabolic (14C-I-glucose oxidation and protein iodination) and chemotactic properties of both cell types were equivalent in suspensions equalized for granulocyte content. These findings indicate that CFFL technique employed does not impair granulocyte viability or function in vitro. Studies of the in vivo survival and function of CFFL granulocytes are necessary to evaluate their efficacy in combating infection in severely leukopenic patients.

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Contribution of LFA-1 and Mac-1 to CD18-dependent neutrophil emigration in a neonatal rabbit model

Journal of Applied Physiology ◽

10.1152/jappl.1996.80.6.1984 ◽

1996 ◽

Vol 80 (6) ◽

pp. 1984-1992 ◽

Cited By ~ 15

Author(s):

J. M. Graf ◽

C. W. Smith ◽

M. M. Mariscalco

Keyword(s):

Monoclonal Antibody ◽

Polymorphonuclear Leukocytes ◽

Rabbit Model ◽

Transendothelial Migration ◽

Peritoneal Exudate ◽

Adult Rabbit ◽

Primary Mechanism ◽

Phosphate Buffered Saline

Human neonatal polymorphonuclear leukocytes (PMNs) exhibit decreased mobility, adherence, and transendothelial migration in vitro compared with adult PMNs. These deficits, in part, are due to functional and quantitative defects in neonatal Mac-1 (CD11b/CD18), whereas LFA-1 (CD11a/CD18) function is similar to that found in adults (D.C.Anderson, O.Abbassi, T.K.Kishimoto, J.M.Koenig, L.V.McIntire, and C.W.Smith, J.Immunol. 146: 3372-3379, 1991; C. W. Smith, T. K. Kishimoto, O. Abbassi, B.J.Hughes, R.Rothlein, L.V.McIntire, E.Butcher, and D.C. Anderson, J. Clin. Invest. 87: 609-618, 1991). We tested the hypothesis that the primary mechanism for the neonatal PMN CD18-dependent emigration in vivo is due to LFA-1. Neutrophils from 1-day-old rabbit pups had 32 and 60% of adult rabbit levels of CD11a and CD11b, respectively. Rabbit pups or adult rabbits received the monoclonal antibody (MAb) R7.1 (anti-CD11a) or R15.7 (anti-CD18) or the vehicle phosphate-buffered saline (PBS) before the instillation of intraperitoneal thioglycollate. Six hours later peritoneal exudate was collected. The administration of MAbs R7.1 and R15.7 in adult animals resulted in 60 and 83% inhibition of leukocyte emigration, respectively, compared with PBS-treated animals (P < 0.01). In neonatal animals, R7.1 and R15.7 inhibited leukocyte peritoneal accumulation to the same extent (50 and 60%, respectively) compared with PBS-treated animals (P < 0.01). Adult animals were also treated with the anti-CD11b MAb 198. MAb 198 decreased emigration by 25%, although this was not significant compared with PBS-treated animals. We conclude that although neonatal animals have significantly less neutrophil CD11a, the diminished levels did not contribute to a reduced ability to emigrate to the peritoneum and, like adult animals, neonatal animals primarily utilize LFA-1 for accumulation in this model. The contribution of Mac-1 to neonatal leukocyte emigration remains uncertain.

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Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.00404-20 ◽

2020 ◽

Vol 202 (23) ◽

Author(s):

Anastasiia N. Klimova ◽

Steven J. Sandler

Keyword(s):

Escherichia Coli ◽

Mutational Analysis ◽

Wild Type ◽

Content Type ◽

Growth Defects ◽

C Terminus ◽

K 12 ◽

And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

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The Mouse Inhalation Model ofCryptococcus neoformansInfection Recapitulates Strain Virulence in Humans and Shows that Closely Related Strains Can Possess Differential Virulence

Infection and Immunity ◽

10.1128/iai.00046-19 ◽

2019 ◽

Vol 87 (5) ◽

Cited By ~ 13

Author(s):

Liliane Mukaremera ◽

Tami R. McDonald ◽

Judith N. Nielsen ◽

Christopher J. Molenaar ◽

Andrew Akampurira ◽

...

Keyword(s):

Survival Time ◽

Cryptococcal Meningitis ◽

Strong Association ◽

Human Infection ◽

Sequence Type ◽

Disease Outcome ◽

Content Type ◽

Fungal Burden

ABSTRACTCryptococcal meningitis (CM) causes high rates of HIV-related mortality, yet theCryptococcusfactors influencing patient outcome are not well understood. Pathogen-specific traits, such as the strain genotype and degree of antigen shedding, are associated with the clinical outcome, but the underlying biology remains elusive. In this study, we examined factors determining disease outcome in HIV-infected cryptococcal meningitis patients infected withCryptococcus neoformansstrains with the same multilocus sequence type (MLST). Both patient mortality and survival were observed during infections with the same sequence type. Disease outcome was not associated with the patient CD4 count. Patient mortality was associated with higher cryptococcal antigen levels, the cerebrospinal fluid (CSF) fungal burden by quantitative culture, and low CSF fungal clearance. The virulence of a subset of clinical strains with the same sequence type was analyzed using a mouse inhalation model of cryptococcosis. We showed a strong association between human and mouse mortality rates, demonstrating that the mouse inhalation model recapitulates human infection. Similar to human infection, the ability to multiplyin vivo, demonstrated by a high fungal burden in lung and brain tissues, was associated with mouse mortality. Mouse survival time was not associated with singleC. neoformansvirulence factorsin vitroorin vivo; rather, a trend in survival time correlated with a suite of traits. These observations show that MLST-derived genotype similarities betweenC. neoformansstrains do not necessarily translate into similar virulence either in the mouse model or in human patients. In addition, our results show thatin vitroassays do not fully reproducein vivoconditions that influenceC. neoformansvirulence.

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Engineered anti-CD70 antibody with multiple effector functions exhibits in vitro and in vivo antitumor activities

Blood ◽

10.1182/blood-2006-07-034017 ◽

2006 ◽

Vol 109 (3) ◽

pp. 1185-1192 ◽

Cited By ~ 73

Author(s):

Julie A. McEarchern ◽

Ezogelin Oflazoglu ◽

Leigh Francisco ◽

Charlotte F. McDonagh ◽

Kristine A. Gordon ◽

...

Keyword(s):

Monoclonal Antibody ◽

Hematologic Malignancies ◽

Effector Cells ◽

Effector Functions ◽

Normal Tissues ◽

Non Hodgkin Lymphoma ◽

Human Igg1 ◽

Constant Domains

Abstract Antigens expressed on malignant cells in the absence of significant expression on normal tissues are highly desirable targets for therapeutic antibodies. CD70 is a TNF superfamily member whose normal expression is highly restricted but is aberrantly expressed in hematologic malignancies including non-Hodgkin lymphoma (NHL), Hodgkin disease, and multiple myeloma. In addition, solid tumors such as renal cell carcinoma, nasopharyngeal carcinoma, thymic carcinoma, meduloblastoma, and glioblastoma express high levels of this antigen. To functionally target CD70-expressing cancers, a murine anti-CD70 monoclonal antibody was engineered to contain human IgG1 constant domains. The engineered antibody retained the binding specificity of the murine parent monoclonal antibody and was shown to induce Fc-mediated effector functions including antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis in vitro. Further, administration of this antibody significantly prolonged survival of severe combined immunodeficient (SCID) mice bearing CD70+ disseminated human NHL xenografts. Survival of these mice was dependent upon the activity of resident effector cells including neutrophils, macrophages, and natural killer (NK) cells. These data suggest that an anti-CD70 antibody, when engineered to contain human IgG1 constant domains, possesses effector cell–mediated antitumor activity and has potential utility for anticancer therapy.

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Polymorphonuclear Leukocytes Restrict Growth of Pseudomonas aeruginosa in the Lungs of Cystic Fibrosis Patients

Infection and Immunity ◽

10.1128/iai.01969-14 ◽

2014 ◽

Vol 82 (11) ◽

pp. 4477-4486 ◽

Cited By ~ 88

Author(s):

Kasper N. Kragh ◽

Morten Alhede ◽

Peter Ø. Jensen ◽

Claus Moser ◽

Thomas Scheike ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Growth Rates ◽

Polymorphonuclear Leukocytes ◽

Growth Patterns ◽

Tissue Samples ◽

Content Type ◽

Growth Physiology

ABSTRACTCystic fibrosis (CF) patients have increased susceptibility to chronic lung infections byPseudomonas aeruginosa, but the ecophysiology within the CF lung during infections is poorly understood. The aim of this study was to elucidate thein vivogrowth physiology ofP. aeruginosawithin lungs of chronically infected CF patients. A novel, quantitative peptide nucleic acid (PNA) fluorescencein situhybridization (PNA-FISH)-based method was used to estimate thein vivogrowth rates ofP. aeruginosadirectly in lung tissue samples from CF patients and the growth rates ofP. aeruginosain infected lungs in a mouse model. The growth rate ofP. aeruginosawithin CF lungs did not correlate with the dimensions of bacterial aggregates but showed an inverse correlation to the concentration of polymorphonuclear leukocytes (PMNs) surrounding the bacteria. A growth-limiting effect onP. aeruginosaby PMNs was also observedin vitro, where this limitation was alleviated in the presence of the alternative electron acceptor nitrate. The finding thatP. aeruginosagrowth patterns correlate with the number of surrounding PMNs points to a bacteriostatic effect by PMNs via their strong O2consumption, which slows the growth ofP. aeruginosain infected CF lungs. In support of this, the growth ofP. aeruginosawas significantly higher in the respiratory airways than in the conducting airways of mice. These results indicate a complex host-pathogen interaction in chronicP. aeruginosainfection of the CF lung whereby PMNs slow the growth of the bacteria and render them less susceptible to antibiotic treatment while enabling them to persist by anaerobic respiration.

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Preclinical Efficacy of a Lipooligosaccharide Peptide Mimic Candidate Gonococcal Vaccine

mBio ◽

10.1128/mbio.02552-19 ◽

2019 ◽

Vol 10 (6) ◽

Cited By ~ 3

Author(s):

Sunita Gulati ◽

Michael W. Pennington ◽

Andrzej Czerwinski ◽

Darrick Carter ◽

Bo Zheng ◽

...

Keyword(s):

Monoclonal Antibody ◽

Neisseria Gonorrhoeae ◽

Vaccine Candidate ◽

Peptide Vaccine ◽

Human Infection ◽

Effective Vaccine ◽

Infection Model ◽

Content Type ◽

Peptide Mimic

ABSTRACT 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. IMPORTANCE Neisseria 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.

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Haemophilus ducreyi Seeks Alternative Carbon Sources and Adapts to Nutrient Stress and Anaerobiosis during Experimental Infection of Human Volunteers

Infection and Immunity ◽

10.1128/iai.00048-16 ◽

2016 ◽

Vol 84 (5) ◽

pp. 1514-1525 ◽

Cited By ~ 6

Author(s):

Dharanesh Gangaiah ◽

Xinjun Zhang ◽

Beth Baker ◽

Kate R. Fortney ◽

Hongyu Gao ◽

...

Keyword(s):

Transmitted Disease ◽

Dna Recombination ◽

Human Infection ◽

Nutrient Stress ◽

Haemophilus Ducreyi ◽

Differentially Expressed ◽

Virulence Determinants ◽

Content Type

Haemophilus ducreyicauses the sexually transmitted disease chancroid in adults and cutaneous ulcers in children. In humans,H. ducreyiresides in an abscess and must adapt to a variety of stresses. Previous studies (D. Gangaiah, M. Labandeira-Rey, X. Zhang, K. R. Fortney, S. Ellinger, B. Zwickl, B. Baker, Y. Liu, D. M. Janowicz, B. P. Katz, C. A. Brautigam, R. S. Munson, Jr., E. J. Hansen, and S. M. Spinola, mBio 5:e01081-13, 2014,http://dx.doi.org/10.1128/mBio.01081-13) suggested thatH. ducreyiencounters growth conditions in human lesions resembling those found in stationary phase. However, howH. ducreyitranscriptionally responds to stress during human infection is unknown. Here, we determined theH. ducreyitranscriptome in biopsy specimens of human lesions and compared it to the transcriptomes of bacteria grown to mid-log, transition, and stationary phases. Multidimensional scaling showed that thein vivotranscriptome is distinct from those ofin vitrogrowth. Compared to the inoculum (mid-log-phase bacteria),H. ducreyiharvested from pustules differentially expressed ∼93 genes, of which 62 were upregulated. The upregulated genes encode homologs of proteins involved in nutrient transport, alternative carbon pathways (l-ascorbate utilization and metabolism), growth arrest response, heat shock response, DNA recombination, and anaerobiosis.H. ducreyiupregulated few genes (hgbA,flp-tad, andlspB-lspA2) encoding virulence determinants required for human infection. Most genes regulated by CpxRA, RpoE, Hfq, (p)ppGpp, and DksA, which control the expression of virulence determinants and adaptation to a variety of stresses, were not differentially expressedin vivo, suggesting that these systems are cycling on and off during infection. Taken together, these data suggest that thein vivotranscriptome is distinct from those ofin vitrogrowth and that adaptation to nutrient stress and anaerobiosis is crucial forH. ducreyisurvival in humans.

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

mBio ◽

10.1128/mbio.00242-21 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Elizabeth M. Parzych ◽

Sunita Gulati ◽

Bo Zheng ◽

Mamadou A. Bah ◽

Sarah T. C. Elliott ◽

...

Keyword(s):

Monoclonal Antibody ◽

Neisseria Gonorrhoeae ◽

Bactericidal Activity ◽

Complement Activation ◽

Natural Infection ◽

Effective Vaccine ◽

Content Type ◽

Synthetic Dna ◽

Human Igg1

ABSTRACT 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. IMPORTANCE Neisseria 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.

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