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Antibody to a conserved antigenic target is protective against diverse prokaryotic and eukaryotic pathogens

Colette Cywes-Bentley, David Skurnik, Tanweer Zaidi, Damien Roux, Rosane B. DeOliveira, Wendy S. Garrett, Xi Lu, Jennifer O’Malley, Kathryn Kinzel, Tauqeer Zaidi, Astrid Rey, Christophe Perrin, Raina N. Fichorova, Alexander K. K. Kayatani, Tomas Maira-Litràn, Marina L. Gening, Yury E. Tsvetkov, Nikolay E. Nifantiev, Lauren O. Bakaletz, Stephen I. Pelton, Douglas T. Golenbock, and Gerald B. Pier
PNAS June 11, 2013 110 (24) E2209-E2218; https://doi.org/10.1073/pnas.1303573110
Colette Cywes-Bentley
aDivision of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital/Harvard Medical School, Boston, MA 02115;
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David Skurnik
aDivision of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital/Harvard Medical School, Boston, MA 02115;
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Tanweer Zaidi
aDivision of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital/Harvard Medical School, Boston, MA 02115;
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Damien Roux
aDivision of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital/Harvard Medical School, Boston, MA 02115;
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Rosane B. DeOliveira
bDepartment of Medicine, University of Massachusetts Medical School, Worcester, MA 01605;
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Wendy S. Garrett
cDepartments of Immunology and Infectious Diseases, Genetics and Complex Diseases, Dana–Farber Cancer Institute, Harvard School of Public Health, Boston, MA 02115;
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Xi Lu
aDivision of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital/Harvard Medical School, Boston, MA 02115;
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Jennifer O’Malley
aDivision of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital/Harvard Medical School, Boston, MA 02115;
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Kathryn Kinzel
aDivision of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital/Harvard Medical School, Boston, MA 02115;
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Tauqeer Zaidi
aDivision of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital/Harvard Medical School, Boston, MA 02115;
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Astrid Rey
dSanofi Research and Development, Therapeutic Strategic Unit, Infectious Disease, 31270 Toulouse, France;
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Christophe Perrin
dSanofi Research and Development, Therapeutic Strategic Unit, Infectious Disease, 31270 Toulouse, France;
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Raina N. Fichorova
eLaboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital/Harvard Medical School, Boston, MA 02115;
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Alexander K. K. Kayatani
fVaccine Branch, Military Malaria Research Program, Walter Reed Army Institute of Research, Silver Spring, MD 20910;
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Tomas Maira-Litràn
aDivision of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital/Harvard Medical School, Boston, MA 02115;
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Marina L. Gening
gLaboratory of Glycoconjugate Chemistry, N. D. Zelinsky Institute of Organic Chemistry, Moscow 119991, Russia;
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Yury E. Tsvetkov
gLaboratory of Glycoconjugate Chemistry, N. D. Zelinsky Institute of Organic Chemistry, Moscow 119991, Russia;
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Nikolay E. Nifantiev
gLaboratory of Glycoconjugate Chemistry, N. D. Zelinsky Institute of Organic Chemistry, Moscow 119991, Russia;
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Lauren O. Bakaletz
hThe Research Institute at Nationwide Children's Hospital and The Ohio State University College of Medicine, Columbus, OH 43205; and
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Stephen I. Pelton
iDepartment of Pediatric Infectious Diseases, Boston University Medical Center, Boston, MA 02118
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Douglas T. Golenbock
bDepartment of Medicine, University of Massachusetts Medical School, Worcester, MA 01605;
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Gerald B. Pier
aDivision of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital/Harvard Medical School, Boston, MA 02115;
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  • For correspondence: gpier@rics.bwh.harvard.edu
  1. Edited* by John J. Mekalanos, Harvard Medical School, Boston, MA, and approved April 24, 2013 (received for review February 26, 2013)

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Significance

Poly-N-acetylglucosamine (PNAG) has been identified as a conserved surface polysaccharide produced by major bacterial, fungal, and protozoal parasites, including malarial sporozoites and blood-stage forms, which can all be targeted for vaccination using this single antigen. Surface carbohydrates are among the most successful vaccines against human microbial pathogens but have tremendous variability that complicates vaccine development. The species of bacteria, fungi, and protozoa shown here to produce PNAG lack an identifiable genetic locus for this antigen’s biosynthetic proteins based on known loci, indicative of a possible evolutionary convergent acquisition of PNAG synthesis with potential important significance for microbial biology.

Abstract

Microbial capsular antigens are effective vaccines but are chemically and immunologically diverse, resulting in a major barrier to their use against multiple pathogens. A β-(1→6)–linked poly-N-acetyl-d-glucosamine (PNAG) surface capsule is synthesized by four proteins encoded in genetic loci designated intercellular adhesion in Staphylococcus aureus or polyglucosamine in selected Gram-negative bacterial pathogens. We report that many microbial pathogens lacking an identifiable intercellular adhesion or polyglucosamine locus produce PNAG, including Gram-positive, Gram-negative, and fungal pathogens, as well as protozoa, e.g., Trichomonas vaginalis, Plasmodium berghei, and sporozoites and blood-stage forms of Plasmodium falciparum. Natural antibody to PNAG is common in humans and animals and binds primarily to the highly acetylated glycoform of PNAG but is not protective against infection due to lack of deposition of complement opsonins. Polyclonal animal antibody raised to deacetylated glycoforms of PNAG and a fully human IgG1 monoclonal antibody that both bind to native and deacetylated glycoforms of PNAG mediated complement-dependent opsonic or bactericidal killing and protected mice against local and/or systemic infections by Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, Neisseria meningitidis serogroup B, Candida albicans, and P. berghei ANKA, and against colonic pathology in a model of infectious colitis. PNAG is also a capsular polysaccharide for Neisseria gonorrhoeae and nontypable Hemophilus influenzae, and protects cells from environmental stress. Vaccination targeting PNAG could contribute to immunity against serious and diverse prokaryotic and eukaryotic pathogens, and the conserved production of PNAG suggests that it is a critical factor in microbial biology.

  • immunotherapy
  • infectious diseases
  • malaria
  • carbohydrates
  • animal models

Vaccination was a major contributor to the success in the 20th century of adding 29.2 y to life expectancy in the United States (1) via a substantial contribution to the control of infectious diseases. However, further success remains challenging in the 21st century due to the appearance of new infectious agents such as severe acute respiratory syndrome and pandemic influenza strains; the reemergence of tuberculosis (TB); and the appearance of multiple antibiotic-resistant organisms. Unfortunately, development of new vaccines is fraught with numerous challenges, among which one of the greatest is the large chemical, and hence immunologic, diversity of protective antigens. Discovery and use of single vaccine components eliciting broadly neutralizing immune effectors similar to the antibodies being sought for HIV and influenza infections (2, 3) would represent a major means to enhance human and animal health.

For bacterial pathogens, surface capsules, often composed of highly varied carbohydrate polymers, have been used successfully to vaccinate humans against Streptococcus pneumoniae (4), Hemophilus influenzae type b (5), Neisseria meningitidis (6), and Salmonella enterica serovar typhi (7). However, efficacy is limited to the capsule types included in the vaccines. Over the past 16 y, the occurrence of a β-(1→6)–linked polymeric-N-acetylglucosamine (PNAG) polysaccharide has been discovered as a component of the bacterial surfaces of a limited number of species (8⇓⇓⇓⇓⇓–14). In these organisms, PNAG production is dependent on the presence of a four-gene locus encoding its biosynthetic enzymes, termed intercellular adhesion (ica) in Staphylococcus (15) and polyglucosamine (pga) or hemin storage (hms) in some Gram-negative pathogens (10). Notably, among seven distinct bacterial species containing these different loci, PNAG has been isolated and demonstrated to be chemically identical with only small variations in acetylation levels of the amino groups and variations in the amount of O-linked acetates and succinates (8, 10, 12, 16⇓–18).

Natural antibody to PNAG is common in humans, rabbits, and goats, but does not elicit robust opsonic killing or immune protection against PNAG-producing bacteria (19, 20) due to inadequate engagement of the serum complement cofactor system needed for optimal protective immunity (21). Thus, broad-based immunity to this antigen has not developed naturally in human or animal populations because PNAG does not usually induce effective antibody responses via microbial colonization or infection (17, 19, 20, 22). Conveniently, chemical alteration of the PNAG molecule to reduce the acetylation level on the glucosamine monosaccharides gives rise to a poly-d-glucosamine glycoform, termed deacetylated PNAG (dPNAG), which elicits antibodies with excellent engagement of the complement system. These antibodies have robust in vitro killing activity and protection against infection by ica/pga-positive pathogens in experimental animals (17, 23). Protective immunity can also be elicited by synthetic oligomers of β-(1→6)–linked glucosamine (GlcNH2), but not synthetic oligo-β-(1→6)–linked N-acetylglucosamines (GlcNAc), conjugated to carrier proteins (22) as well as by administration of a fully human IgG1 monoclonal antibody (mAb) that binds to both PNAG and dPNAG and deposits complement component C3 onto the bacterial surface (21).

The synthesis of PNAG by both Gram-positive and Gram-negative pathogens (8, 10, 12⇓–14) suggests an important role for this molecule in microbial biology. However, analyses of many published genomes fail to identify genetic loci similar to the known four-gene ica/pga loci (10, 15). We hypothesized that in ica/pga-negative organisms PNAG could be produced by biosynthetic enzymes encoded in related but currently unidentifiable genetic loci. Therefore, if PNAG is broadly distributed among human pathogens it could serve as a vaccine antigen targeting many different microbes.

To evaluate this hypothesis, we developed a definitive means to detect PNAG production by sensitive and specific immunochemical analysis, necessitated by the failure, after extensive attempts, at chemical characterizations using GC/MS or NMR on preparations obtained from many microbes. Additional information for the basis for this failure is presented in SI Text 1. The robust and specific immunochemical fluorescence analysis we developed to detect PNAG on multiple organisms was based on binding to microbial surfaces of both a fully human IgG1 mAb and polyclonal antibody to PNAG raised in four different animal species. All of these different antibodies bind to organisms with known PNAG biosynthetic loci and have no reactivity to isogenic mutants that have these loci deleted (13, 14, 17, 21, 22, 24⇓⇓⇓–28). Recovery of antibody binding is achieved when an intact ica/pga locus is introduced into the mutants. To confirm that the antibody we used to detect PNAG was binding to a β-(1→6)–linked N-acetylglucosamine polymer, we digested bacterial samples with the extensively characterized enzyme dispersin B (12, 29, 30), whose crystal structure (31), active site residues (29), and substrate specificity (30) show this enzyme can only cleave β-(1→6)–linked glucosamines. Samples were also treated with sodium metaperiodate, which can only degrade β-(1→6)–linked glucosamine residues and not any other possible linkages available for glucosamine polymers. From these multiple analyses we found not only that many major human bacterial pathogens produce PNAG, but also eukaryotic pathogens, including important fungi, the human protozoan parasite Trichomonas vaginalis, and the rodent and human apicomplexan malarial parasites Plasmodium berghei ANKA and Plasmodium falciparum. Antibody to PNAG-mediated opsonic/bactericidal killing of a representative group of Gram-positive and Gram-negative bacteria and fungi provided protection against this diverse set of pathogens, including cerebral malaria in mice, indicative of the potential of PNAG to serve as a unique and broad-spectrum vaccine eliciting immunity against a wide range of major human and animal pathogens.

Results

Occurrence of PNAG Among Diverse Microbial Pathogens.

PNAG was detected by confocal microscopy on the surface of microbial cells using the antigen-specific human IgG1 mAb F598 (Fig. S1A) (21) directly conjugated to Alexa Fluor 488 (AF488, green fluorescence). The specificity of the mAb is shown by binding to wild-type Staphylococcus aureus but not to this organism when the ica biosynthetic genes for PNAG are deleted, with binding restored when an intact ica locus is placed in the chromosome (Fig. S1A). The control was a human IgG1 mAb, F429, also directly conjugated to AF488, made in the same IgG1 heavy-chain/lambda light-chain expression vector, and thus having the identical antibody constant regions but with variable regions providing specific binding to the alginate antigen from Pseudomonas aeruginosa (32), an organism in which we are unable to detect PNAG (Fig. S1A). Microbial DNA was detected by staining with SYTO 83 (red fluorescence). As shown in Fig. 1, in addition to the known PNAG-positive control, S. aureus, fluorescence was seen using mAb F598 to PNAG, but not with mAb F429, with the Gram-positive cocci S. pneumoniae (two strains), Streptococcus pyogenes (group A streptococci, or GAS), Streptococcus dysgalactiae (group C strep), and Enterococcus faecalis; Gram-positive rods Listeria monocytogenes, Clostridium difficile, Mycobacterium tuberculosis, and Mycobacterium smegmatis; the Gram-negative cocci and coccobacilli N. meningitidis, Neisseria gonorrheae, nontypable H. influenzae, Haemophilus ducreyi, Helicobacter pylori, and Campylobacter jejuni; and the Gram-negative rods Citrobacter rodentium and S. enterica serovars typhi and typhimurium. Notably, the last three organisms are closely related to E. coli but lack a readily identifiable four-gene pga locus. Unexpectedly, we also found mAb F598 bound to yeast and hyphal forms of Candida albicans, as well as to the fungal organisms Aspergillus flavus, Fusarium solani, and Cryptococcus neoformans. An ELISA inhibition test was used to ascertain that mAb F598 was not binding to fungal glucans (Fig. S1B). mAb F598 also bound to T. vaginalis cells, an obligatory extracellular parasite of the human genitourinary tract, which contains β-GlcNAc in its surface lipophosphoglycan structure (33) and to P. berghei ANKA and P. falciparum parasites growing inside of mouse or human red blood cells, respectively (Fig. 1). Antibody to PNAG also bound to P. falciparum sporozoites (Fig. S1D). In addition to the results with the strains shown in Fig. 1, other strains of some of these organisms (Table S1) were found to be positive for PNAG production, with some PNAG-negative strains also noted (Table S1).

Fig. 1.
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Fig. 1.

Expression of PNAG among diverse microbial pathogens. Cells from cultures of the indicated microbes were fixed with paraformaldehyde, placed onto slides, exposed to cold methanol, and then reacted with either control mAb F429 conjugated to AF488 or mAb F598 to PNAG also conjugated to AF488 (green fluorescence) as well as with SYTO 83 to visualize DNA (red fluorescence) and photographed using a scanning confocal laser microscopy. The label “dual” indicates overlay of red and green channels. (Scale bars: 10 µm; note that some micrographs lack bars due to cropping but are at same magnification as other micrographs of same strain.)

Confirmation that mAb F598 was binding to PNAG or a closely related surface oligosaccharide that would need to be at least seven N-acetylglucosamine monomers in length (Fig. S1C) was obtained by subjecting cells to digestion with the PNAG-degrading enzyme dispersin B (12) that specifically hydrolyzes β-(1→6)–linked N-acetylglucosamine (29, 30); a related degradative control enzyme, chitinase, that specifically hydrolyzes the β-(1→4)–linked N-acetylglucosamine chitin molecule; and treatment with sodium metaperiodate. Chitinase-resistant but dispersin B- and periodate-sensitive binding of mAb F598 would thus be highly specific to PNAG or PNAG oligosaccharides. All of the organisms tested met these immunochemical criteria (see Fig. S1E for P. falciparum sporozoites and Fig. S2 for other organisms).

S. pneumoniae is well known for having >90 variable capsular polysaccharide serotypes. Thus, current vaccine formulations contain from seven to 23 different conjugated or purified polysaccharide components (34). Highly effective protection against systemic pneumococcal disease has been achieved in children by use of conjugate vaccines containing 7, 10, or 13 distinct conjugated capsular polysaccharides (34), but the use of these vaccines has raised concern that selection for nonvaccine serotypes will result in them becoming more common causes of infection. To ascertain if PNAG was a shared surface polysaccharide by multiple strains of S. pneumoniae, we analyzed all 13 strains contained in the most comprehensive conjugate vaccine and an additional eight nonvaccine strains, and found all 21 had detectable surface PNAG (Fig. S3), suggesting that a PNAG-targeted vaccine may effectively prevent infection by a large number of pneumococcal strains.

PNAG and Encapsulation of N. gonorrheae, N. meningitidis Serogroup B, and H. influenzae.

Because both N. gonorrheae and nontypable H. influenzae are extensively studied bacterial pathogens that to date have not been found to produce a capsule, we choose these two special examples to ascertain that PNAG is, in fact, their capsular polysaccharide. Historically, bacterial capsules have been defined by immunoelectron microscopic detection of the antigen surrounding the bacterial surface bound to specific antibody. A more specific definition has not been substantiated. For three isolates of N. gonorrheae and three clinical H. influenzae isolates that were not typable using polyclonal antibodies to H. influenzae capsular antigens, we could demonstrate, via mAb F598 binding, that PNAG is a capsule-like entity for these organisms (Fig. 2). The mAb F598 also bound to PNAG surrounding N. meningitidis serogroup B, a major pathogen for which a capsule-specific vaccine is not possible due to the self-antigenic, nonimmunogenic properties of the serogroup B antigen. To demonstrate that the PNAG molecules were spatially located in the same area as classic capsular antigens, we also costained serogroup A N. meningitidis with mAb F598 to PNAG and a mouse IgG mAb to the serogroup A antigen, and differentiated these antibodies with protein A bound to 15-nm or 10-nm gold particles, respectively (Fig. 2). The different-sized gold particles were seen mixed together on the bacterial surface, indicating that PNAG and the classic N. meningitidis capsular antigens were intercalated on the bacterial surface.

Fig. 2.
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Fig. 2.

Demonstration that PNAG is a surface capsular polysaccharide for N. gonorrhoeae and nontypable H. influenzae and is also surface expressed on N. meningitidis serogroups B and A. Indicated organism (A–I) was reacted with control mAb F429 to P. aeruginosa alginate or mAb F598 to PNAG followed by protein A conjugated to 15-nM gold particles. (J) N. meningitidis serogroup A strain Z2087 cells were first reacted with either control mAb F429 or mAb F598 to PNAG then with 15-nm protein A gold particles followed by 1% glutaraldehyde to cross-link the human IgG1 mAbs and the 15-nm protein A gold label and to block further binding of protein A. Next, a mouse IgG mAb to the serogroup A capsule was applied to the grids, followed by a bridging rabbit antibody to mouse IgG then by 10-nm protein A gold particles. Control + anti-serogroup A panels show binding only of the antibody to serogroup A, whereas panels labeled anti-PNAG (598) + anti-serogroup A show binding of both antibodies. High magnification of indicated surface area (purple arrow) shows intercalation of selected examples of both 10-nm (purple circles) and 15-nm (green circles) gold particles on the bacterial surface.

Expression of PNAG in Vivo During Human and Animal Infection.

To effectively serve as a vaccine target, PNAG must be expressed on the microbial surface in vivo. We analyzed a variety of human and animal tissues infected with different pathogens for PNAG expression. Among four tested samples of middle ear effusions (MEF) from children with S. pneumoniae otitis media and two MEF samples from children with nontypable H. influenzae otitis media, we detected chitinase-resistant, dispersin B-sensitive PNAG on the infecting bacteria, wherein the bacterial cells were also stained with either a S. pneumoniae or H. influenzae-specific antibody (Fig. 3 A–F). In lung tissue from a M. tuberculosis-infected patient, we detected PNAG on tubercle bacilli (Fig. 3 I–L) physically associated with M. tuberculosis antigens that reacted with a bacterium-specific antibody. Though there was colocalization of PNAG and M. tuberculosis antigens (Fig. 3 J–L), there were also some mycobacterial antigens visualized that were not associated with PNAG (Fig. 3J). We could not ascertain if the mycobacterial cells were intracellular in these sections that also were not amenable to treatment with enzymes or periodate. In nasopharyngeal fluid from chinchillas experimentally infected with S. pneumoniae serogroup 19A, colocalization of the chitinase-resistant, dispersin B-sensitive PNAG antigen with the serogroup 19A capsule was readily seen (Fig. 3 G and H). In the gastrointestinal tract of a mouse experimentally infected with C. rodentium, considered to be the murine equivalent of human enteropathogenic Escherichia coli (35), PNAG was detected around microbes associated with the epithelial cells (Fig. 3M). In ocular tissues from mice with C. albicans keratitis, PNAG was detected on the DNA-positive portion of the fungal cells, but not prominently on the hyphal stalks extending into the tissues (Fig. 3N).

Fig. 3.
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Fig. 3.

In vivo expression of PNAG by various microbial pathogens. Samples of infected middle ear fluid (MEF) from humans with either S. pneumoniae (A–D) or nontypable H. influenzae (E and F) otitis media (OM) or chinchillas with OM due to S. pneumoniae serotype 19A (G and H) were treated with chitinase and reacted with control mAb F429-AF488 to P. aeruginosa alginate or treated with chitinase, dispersin B, or periodate in the case of nontypable H. influenzae (periodate destroyed S. pneumoniae cells) and reacted with mAb F598-AF488 to PNAG. (I) Colonic sections from mice with C. rodentium infection were stained with either control mAb F429-AF488 or mAb F598-AF488 to PNAG (green) plus SYTO 83 (red) to visualize DNA. Two different sections stained for both PNAG and DNA shown. (J) Sections from the cornea of a mouse with C. albicans keratitis stained with either control mAb F429-AF488 or mAb F598-AF488 to PNAG (green) plus SYTO 83 (red) to visualize DNA. Sections of a human lung infected with M. tuberculosis (K–N) were stained with SYTO 62 to visualize DNA (blue), rabbit antibody to M. tuberculosis followed by anti-rabbit IgG secondary antibody (red), or control mAb F429-AF488 or anti PNAG-mAb F598-AF488 (green). (Scale bars: white, 10 µm; red, 20 µM; some micrographs lack bars due to cropping but are at the same magnification as other micrographs of the same strain.)

In Vitro Opsonic and Bactericidal Killing.

We next assessed the ability of polyclonal antibody and/or mAb F598 to mediate opsonic killing of PNAG-producing Gram-positive cocci for which effective capsular polysaccharide vaccines exist but more extensive coverage may be needed (S. pneumoniae) or for which an immunogenic, non–self-antigenic carbohydrate capsule has not been described (E. faecalis and S. pyogenes) and also for the fungal pathogen C. albicans. Additionally, we analyzed phagocyte-independent bactericidal killing of Neisseria spp., all of which were found by confocal microscopic visualization to produce PNAG. These in vitro activities are the main correlates of immunity engendered by effective capsular polysaccharide vaccines (19, 36). Both polyclonal antibody and mAb F598 mediated opsonic killing of four strains of S. pneumoniae (Fig. 4 A and B); polyclonal antibody mediated killing of three distinct E. faecalis strains (Fig. 4C); and mAb F598 mediated killing of two S. pyogenes strains (003 and 771) as well as a mutant of strain 771 lacking the ability to produce the hyaluronic acid group A streptococci (GAS) capsule (strain 188; Fig. 4D). Similarly, mAb F598 mediated opsonic killing of C. albicans (Fig. 4E).

Fig. 4.
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Fig. 4.

Opsonic or bactericidal killing of selected microbial pathogens by antibody to PNAG (anti-9GlcNH2-TT). (A and B) Opsonic killing of four strains of S. pneumoniae mediated by polyclonal rabbit (A) or mAb (B) to PNAG in the presence of HL60 phagocytes and complement (C′). Control for mAb F598 is irrelevant mAb F429. (C) Opsonic killing of three strains of E. faecalis mediated by polyclonal antibody to PNAG. (D) Opsonic killing of two strains (003 and 771) of S. pyogenes mediated by mAb F598 as well as a deletion mutant of strain 771 lacking the ability to produce the self-antigenic hyaluronic acid GAS capsule (strain 188). (E) Opsonic killing of C. albicans by indicated amount of mAb F598 in the presence of polymorphonuclear neutrophils (PMNs) and complement. (F and G) Bactericidal killing of six strains of N. gonorrhoeae (F) and five strains of N. meningitidis serogroup B (G) mediated by polyclonal rabbit antibody to PNAG and its indigenous C′. Killing calculated to subtract out any reductions in cfu counts by a comparable dilution of normal rabbit serum (NRS). Heat-inactivated complement (HI C′) controls for all N. gonorrhoeae strains had greater cfu counts surviving than in control NRS, and are not shown on figure. Bars represent means of triplicates to quadruplicates determined in the same assay; bars below the zero line indicate samples with colony counts greater than the control.

Using immune polyclonal antibody raised to the synthetic β-(1→6)–linked glucosamine oligosaccharide conjugated to tetanus toxoid (9GlcNH2-TT) (22) wherein the indigenous complement served as the source of this mediator of bactericidal activity, 7/7 strains of N. gonorrheae tested were killed (Fig. 4F), as were 5/5 strains of serogroup B N. meningitidis (Fig. 4G). The cfu counts in the experimental tubes were vigorously compared and found to be essentially identical before and after sonication to ensure bacterial killing, and not agglutination, was achieved. Using antigen inhibition studies, we demonstrated a dose-dependent specific inhibition of the bactericidal activity using purified PNAG for N. gonorrheae and serogroup B N. meningitidis (Fig. S4), whereas the control polysaccharide P. aeruginosa alginate had no effect. These results clearly substantiate the specificity of the bactericidal activity of antibody to PNAG against Neisseria.

Protective Efficacy of Antibody to PNAG Against Bacterial Infections.

To demonstrate the broadly protective nature of antibody to PNAG, we conducted passive protection studies against a subset of the antigen-positive pathogens that are associated with infections in a variety of tissues. Prior results have shown that antibodies to PNAG are only protective against microbial cells that produce this antigen and not isogenic mutants in which ica or pga genes have been deleted (13, 14, 21, 22, 27, 28). To validate that immunity induced with a synthetic oligosaccharide–protein conjugate (22) would be effective in settings where active vaccination would be the preferred approach, we passively transferred polyclonal antibodies raised to 9GlcNH2-TT to mice before challenge with organisms representative of those causing vaccine-preventable infections. For infections more likely to be treated by passive immunotherapy, such as those mostly occurring when an additional risk factor is acquired, such as tissue trauma or hospitalization for illness or injury, we injected mice with the human IgG1 F598 mAb (21) or control mAb before challenge.

S. pyogenes (GAS) is a prominent cause of acute skin infections that can cause systemic, potentially lethal, infections. Antisera to 9GlcNH2-TT protected mice against a lethal GAS infection initiated in the skin (Fig. 5A). L. monocytogenes is often a cause of infections in neonates (37), and passive administration of antibody to 9GlcNH2-TT protected 100% of 3-d-old neonatal mice against systemic infection (Fig. 5B). To evaluate the protective efficacy of antibody to PNAG against nonvaccine serotypes of S. pneumoniae, we infected CBA/N mice, which are hypersusceptible to pneumococcal infection due to a B-cell development defect and an inability to produce natural protective antibody (38), with serotype 2 strain D39 delivered intranasally into the lungs. Antibody to PNAG significantly reduced the mortality associated with pneumonia and systemic spread (Fig. 5C). In addition, because passive vaccination for nosocomial pneumonia due to S. pneumoniae is also a likely therapeutic target, mAb F598 was given 4 h before intranasal infection with serotype 9V DSM 11865 strain in FVB mice and shown to be as potent as the antibiotic cefotaxime (administered at 1 and 4 h postinfection) in reducing bacterial burdens in the mouse lungs (Fig. 5D).

Fig. 5.
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Fig. 5.

Protective efficacy of antibody to PNAG against experimental mouse infections caused by various pathogens. (A) Protection against lethality due to S. pyogenes (group A Streptococcus) infection initiated from an s.c. injection. (B) Protection against lethal sepsis in 3-d-old neonatal mice following i.p. injection of L. monocytogenes. (C) Protection against lethal, systemic infection in CBA/N mice infected intranasally with S. pneumoniae serotype 2 strain D39. P values for A–C determined by log-rank tests. (D) Reductions in bacterial burden 24 h after infection into the lungs of FVB mice given indicated dose of mAb F598 i.v. 4 h before intranasal infection with S. pneumoniae serotype 9V strain. Comparison group given the antibiotic cefotaxime (15 mg/kg) 1 and 4 h postinfection. P values determined by ANOVA (overall P < 0.001) and post hoc, pairwise comparisons made to the control. An irrelevant human IgG1 mAb had no effect on S. pneumoniae clearance in this model (not depicted). (E and F) Levels of N. meningitidis serogroup B strain B16B6 in the brains of 3-d-old mice given 50 µL of indicated antiserum 24 h before i.p. infection with two different doses of bacteria. Brain levels determined 24 h postinfection. P values determined by nonparametric t test. (G) Cumulative histopathologic scores in TRUC mice at 8 wk of age following treatment commenced in week 1 with either control human IgG1 mAb F105 or mAb F598 to PNAG. (H) Cumulative histopathologic scores in WT mice cross-fostered on TRUC females that transmit infectious colitis to the nursed animals by 8 wk of age. Treatment commenced in week 4 with either control human IgG1 mAb F105 or mAb F598 to PNAG. P values for G and H determined by nonparametric t test. (E–H) Symbols represent individual mice; lines represent median for the group.

Developing vaccines against serogroup B N. meningitidis has been challenging because the capsule of this organism is nonimmunogenic in humans due to chemical identity to a self-antigen (39). We tested the ability of antibody to 9GlcNH2-TT administered 24 h before infection to reduce the levels of N. meningitidis serogroup B entering the brains of 3-d-old neonatal mice 24 h following i.p. infection. At both a low (5 × 105 cfu per mouse) and high (5 × 108 cfu per mouse) challenge dose, polyclonal antibody to PNAG significantly reduced the bacterial levels in the brains (Fig. 5 E and F).

To show a positive effect of mAb F598 in a setting of infectious colitis caused by the indigenous microbial constituents of the murine gastrointestinal tract, and thus unmanipulated in regard to the presence of PNAG-producing organisms, we used the murine BALB/c T-bet−/− RAG2−/− (TRUC) model of spontaneous colitis (40, 41). Pups born to female TRUC mice that lack both the T-bet transcription factor needed for development of innate and adaptive immunity, as well as the recombinase-activating enzyme needed for B and T-cell maturation, develop significant colitis by 8 wk of age that is amenable to treatment by antibiotics (41). In comparison with controls injected with human IgG1 mAb F105 specific to HIV gp120, chosen for the very low likelihood that it would bind to microbes in the indigenous murine gastrointestinal flora, weekly administration of mAb F598 starting at day 7 of life significantly reduced the total histopathologic damage determined at 8 wk of age that was quantified in a blinded fashion by a pathologist (Fig. 5G), encompassing notable reductions in the individual levels of hyperplasia, injury, and polymorphonuclear leukocytes (PMN) infiltration (Fig. S5A). Critically, eight of 10 mAb F598-treated mice had an injury score of zero, whereas six of eight control mice had injury scores of 1–3 (P = 0.029, one-tailed Fisher’s exact test).

When WT neonates are cross-fostered by TRUC females they develop spontaneous colitis at 8 wk (40), although it is less severe than in TRUC offspring. To evaluate the therapeutic potential of mAb F598 against colitis in a setting of unperturbed immune system function, we initiated treatment of WT mice fostered by TRUC females at 4 wk of age with biweekly injections of mAb F598 or control human IgG1 mAb F105 and determined the level of colonic pathology at 8 wk of age. mAb F598 significantly reduced the total pathology score in the recipient mice compared with controls (Fig. 5H), with significant reductions in monocyte infiltration and reactive hyperplasia (Fig. S5B), but not injury, because most of the controls had injury scores of zero.

Protective Efficacy of Antibody to PNAG Against Eukaryotic Microbial Infection.

Because we detected PNAG production by fungal and malarial eukaryotic parasites, we evaluated antibody-mediated protection against C. albicans corneal keratitis and cerebral malaria and death due to systemic P. berghei ANKA infection in mice. Injecting mAb F598 i.p. 24 h before infection of a scratch-injured mouse eye with a low (1 × 105 yeast cells per eye) dose of C. albicans followed by topical application of mAb at 5 µg per eye at 24 and 32 h postinfection significantly reduced the fungal levels (Fig. 6A) and associated corneal pathology (Fig. S5C) at 48 h compared with mice given the control mAb. Notably, mAb treatment cleared detectable infection from the eyes of seven of eight mice challenged with the dose of 1 × 105 yeast cells per eye compared with measurable fungal infections in eight of eight eyes from control Mab-treated mice (P = 0.0007, one-tailed Fisher’s exact test). Using a higher fungal challenge dose (5 × 107 yeast cells per eye) and only topical treatment with 5 µg mAb per eye at 4, 8, and 24 h postinfection, we also obtained a significant reduction in infectious yeast cells (Fig. 6B) and reduced the associated corneal pathology (Fig. S5D) at 32 h when the experiment was terminated in accordance with our approved end-points.

Fig. 6.
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Fig. 6.

Protective efficacy of mAb or polyclonal sera to PNAG against eukaryotic pathogens. (A) Reductions in cfu counts recovered from the cornea of C57BL/6 mice infected with C. albicans during keratitis. Mice treated with 200 µg i.p. of either control IgG1 mAb or F598 24 h before eye infection followed by topical application of 5 µg mAb per eye 24 and 32 h postinfection. Corneas recovered and processed after 48 h of infection. P value by nonparametric t test. (B) Reductions in cfu counts recovered from the cornea of mice infected with C. albicans during keratitis. Mice treated with either control IgG1 mAb or F598 by topical application of 5 µg per eye 4, 8, and 24 h postinfection with corneas recovered for processing 32 h postinfection. P value by nonparametric t test. Corneal pathology scores associated with these treatments are in Fig. S5. Symbols represent individual mice; lines represent the group medians. (C) Survival of C57BL/6 mice from systemic PNAG-positive P. berghei ANKA infection given i.p. injections of either 0.2 mL of normal goat serum or 0.2 mL of goat antibody raised to 9GlcNH2-TT on days −1, +2, +5, +8, +11, +14, +17, and +20. P value by log-rank test. (D) Lack of survival of C57BL/6 mice from systemic PNAG-negative, GFP-positive P. berghei ANKA infection given i.p. injections of either 0.2 mL of normal goat serum or 0.2 mL of goat antibody raised to 9GlcNH2-TT on days −1, +2, +5, and +8.

An effective vaccine for malaria will likely require multiple parasite antigens, but as an initial step in determining if PNAG might be a candidate vaccine antigen component for a multivalent vaccine, we treated C57BL/6 mice infected with PNAG-positive P. berghei ANKA with 200 µL of polyclonal antibody to PNAG or control normal serum injected i.p. every 3 d starting the day before infection and through 20 d postinfection. Antibody to PNAG significantly extended the survival of the treated mice (Fig. 6C) and prevented development of cerebral malaria (Fig. S6A). Five of eight mice treated with normal serum died by day 9 with low levels of parasitemia (Fig. S6B), and the remaining three died by day 30 with high levels of parasitemia. For the mice treated with antibody to PNAG for 3 wk, only one died of cerebral malaria by day 7, four developed increasing levels of parasitemia (Fig. S6C) and died by day 33, which was 13 d after the last injection of antibody, one had no detectable parasitemia until day 22 and died at day 40 (20 d after the last injection of antibody), and two mice had little to no detectable parasitemia and survived the 45-d experimental period (Fig. S6C). Antibody treatment was stopped at day 20 as per the initial protocol stipulation, but we speculate that extended survival might be observed by increasing the duration of antibody treatment.

Unexpectedly, when we tested a variant of P. berghei ANKA expressing GFP (42) for PNAG production, this variant was negative for the antigen, whereas the GFP protein could be readily detected by a GFP-specific antibody (Fig. S6E, red). The basis for the loss of PNAG production during introduction of the GFP construct is unknown, but surface polysaccharides can interfere with DNA uptake (43, 44), and thus natural PNAG-negative variants in a microbial population can have an advantage in regard to uptake of exogenous DNA. We took advantage of the lack of PNAG in the GFP derivative strain to test the efficacy of antibody against the PNAG-negative P. berghei variant and found no protective efficacy. All of the mice in both arms of this experiment died by day 10 (Fig. 6D), confirming the specificity of the protection for the PNAG antigen on the PNAG-positive P. berghei.

PNAG Enhances Microbial Resistance to Environmental Stress.

Microbial biofilms, often containing PNAG, are well known to have enhanced survival against a variety of environmental stresses and antimicrobial factors (45). The common expression of PNAG by diverse microbes suggests the polysaccharide might also protect planktonic cells from environmental stress, similar to the resistance to wetting that occurs in Bacillus subtilis biofilm colonies (46) that can be measured by exposure to ethanol. To assess this property of PNAG on planktonic cells, we used three ica/pga-positive strains wherein we could delete then restore, in trans, the ica/pga genetic loci and test their relative resistance to 5% ethanol in water over 48 h, a level that was not lethal to the WT parental strains. WT and complemented strains of Klebsiella pneumoniae, E. coli, and S. aureus all significantly survived exposure to ethanol better than the ica/pga-negative, PNAG-deficient mutants (Fig. S7), suggesting PNAG serves as a type of microbial armor regulating interactions of planktonic cells with the external environment.

Discussion

The common expression of PNAG on the surface of diverse bacterial, fungal, and protozoal cells provides the potential to elicit immunity via a broadly protective vaccine and/or passive antibody therapy to prevent infections due to multiple microbial pathogens as long as they are susceptible to killing by this humoral immune factor. Importantly, we detected in vivo expression of PNAG on microbes infecting human and animal tissues, consistent with prior findings that in vivo infection increases expression of PNAG (9). Whether immunity to PNAG alone is sufficient to provide adequate protection or will need to be part of multivalent vaccines remains to be determined. Nonetheless, the data presented here demonstrated that antibody raised to a synthetic nonasaccharide of β-(1→6)–linked glucosamine conjugated to TT, or a human IgG1 mAb that binds to both native and dPNAG, detected PNAG expression in vivo, mediated killing of diverse microbial and fungal pathogens, and protected mice against bacterial, fungal, and plasmodial infections. The specificity of these antibodies has previously been demonstrated among bacterial isolates with known ica or pga operons, wherein strains either lacking or deleted for the PNAG biosynthetic loci no longer bound any of the PNAG-specific antibodies, and no in vivo protection was achieved in the absence of PNAG (13, 14, 22, 27, 28). Our studies here extend the documentation of the specificity of this protective immunity to an important eukaryotic pathogen wherein protection against PNAG-positive P. berghei ANKA was lost when mice were challenged with a GFP-variant not expressing PNAG. Although PNAG may not be a virulence factor for cerebral malaria induced in mice by direct injection of parasites, this observation does not exclude the possibility that PNAG expressed on malarial sporozoites might be critical for infectivity via the natural mosquito transmission route.

Although the loss of immunoreactivity following treatment with dispersin B and periodate provide compelling evidence for the presence of an oligosaccharide or polymer of β-(1→6)–linked N-acetylglucosamine on the surface of the microbes we studied, such a result does not completely exclude the possibility that the antigens we detected might be structural variants of the PNAG molecule or, less likely, chemically distinct from PNAG. Even if there are other molecules susceptible to dispersin B and periodate degradation, the immunologic cross-reactivity evident by strong antibody binding, killing, and protection should nonetheless result in a highly effective, broad-spectrum vaccine against numerous pathogens (8, 10, 12, 13, 17, 24, 47⇓–49). The key property of antibodies that bind to PNAG needed for broad protection is either facilitation of fragment crystallizable-mediated immune clearance or, as found for all of the microbes studied to date, deposition of opsonically active or bactericidal complement components onto microbial surfaces.

Our findings that a diverse range of microbes produce surface PNAG or a closely related structure challenges critical paradigms of microbial pathogenesis and immunity—notably, well-established findings that surface carbohydrates tend to be highly diverse to avoid host innate and adaptive immune responses. Despite the heterodoxy proposed here, if additional genomic and biochemical studies support the occurrence of diverse PNAG synthetic enzyme systems among microbes, particularly nonpathogens, the importance of this molecule in microbial biology should be validated. For example, if PNAG is commonly expressed among nonpathogenic microbes, such as the recently estimated 2.9 × 1029 cells within the subseafloor sediments (50), then its production might rival that of other major important biologic polysaccharides with which it shares structural similarity, including chitin, fungal glucans, and potentially even cellulose.

Attempts to identify four-gene biosynthetic loci homologous to the known icaADBC/pgaABCD loci (10, 13, 15, 24) in the genomes of the pathogens studied here were frustrated by the occurrence of large numbers (10–75) of genes and proteins with high homologies to the four known glycosyl transferases: icaA, icaC, pgaA, and pgaC; this made it difficult to determine which ones would be prime candidates that encode a transferase involved in PNAG synthesis among the organisms newly identified as producing PNAG. The glycosyl transferase genes in the genomes of the organisms we studied were also never found in an operon or even in close proximity to genes having homology to either a deacetylase (icaB/pgaB) or to the icaD/pgaD genes whose protein products facilitate IcaA and PgaC polymerization of UDP-N-acetylglucosamine (51, 52). Ultimately, we expect that homologous genes and proteins with alternative genomic organizations and amino acid sequences, respectively, will be identified that are responsible for PNAG synthesis in diverse organisms.

In regards to clinical development of PNAG-based immunotherapies and vaccines for humans, mAb F598 has been successfully tested in a phase I clinical trial for safety and pharmacokinetics (53). No significant adverse events were reported following injection of up to 17 mg/kg of mAb F598 into humans (53). Preclinical tests of a GMP-level mAb F598 preparation found no binding to 33 different tissues from three humans and a Macaca fascicularis monkey, and had acceptable safety in standard toxicology tests in mice (53). There are potential concerns about the effect of antibody to PNAG on normal microbial flora, and these are addressed in SI Text 2. In addition to the mAb, a synthetic oligosaccharide conjugate vaccine is being prepared for human testing (22). Overall, preclinical animal and human tests conducted to date indicate administering or inducing via immunization a complement-fixing antibody to PNAG does not lead to clinically significant minor or major adverse events.

Materials and Methods

Microbial Strains and Tissue Samples.

The strains of microbes, their source, suppliers, and method of growth used in this study are listed in Table S1, as are the sources of animal and humans tissue samples used to detect PNAG expression in vivo. The following reagents were obtained through BEI Resources, National Institute of Allergy and Infectious Diseases, National Institutes of Health: S. pneumoniae strains SPEC1, OREP3, OREP4, STREP5, TREP6A, SPEC6B, OREP7F, EMC9V, STREP14, OREP18C, TREP19A, SPEC19F, and EMC23F, and P. berghei (ANKA) GFP.

Antibodies.

Fully human IgG1 mAbs with V-regions encoding specificity for P. aeruginosa alginate, mAb F429 (32) or PNAG (21) cloned into the TCAE 6.1 vector (human lambda-light chain and gamma-1 heavy chain) have been described. Human IgG1 mAb F105 to HIV gp120 was kindly provided by Lisa Cavacini (Beth Israel Deaconess Medical Center, Boston, MA). Goat antibodies raised to either dPNAG or synthetic 9GlcNH2 conjugated to tetanus toxoid were prepared and used as described (22). Antibodies to S. pneumoniae and the S. pneumoniae 19A capsule, M. tuberculosis, and antibody to nontypable H. influenzae were obtained from AbD Serotec, Statens Serum Institut, AbCam, and LifeSpan BioSciences, respectively. Antibody to serogroup A N. meningitidis was kindly provided by Peter Rice (University of Massachusetts Medical School, Worcester, MA).

Antigens.

Purified PNAG was isolated as described (26). Synthetic oligosaccharides of PNAG were prepared as described (22). β-Glucan from yeast was obtained from Calbiochem.

Confocal Microscopy and Immunochemical Detection of PNAG on Microbial Cells.

Microbial samples from broth or agar were fixed by suspension in 4% paraformaldehyde, then spotted onto microscope slides, air-dried, and covered for ∼1 min with ice-cold methanol. After washing, slides were reacted with control or PNAG-specific mAbs directly conjugated to Alexa Fluor 488 at 5.2 µg/mL along with indicated DNA-visualizing dye. After 2 h at room temperature (RT) or overnight at 4 °C, slides were washed and observed by confocal microscopy. For enzymatic and periodate treatments, cell samples or samples already fixed to slides were incubated in Tris-buffered saline (pH 6.4) containing either 50 µg dispersin B per milliliter (digests PNAG antigen) (54) or 50 µg chitinase per milliliter (no effect on PNAG antigen but tested to ensure it was active by treating chitin then measuring the loss of chitin binding to wheat-germ agglutinin) overnight at 37 °C or in 0.4 M periodate (destroys PNAG) for 2 h at 37 °C. After washing, cells were treated with the AF488 directly conjugated mAbs.

Detection of PNAG on P. falciparum Sporozoites.

To prepare sporozoite slides, NF54 mosquitoes were dissected by cutting the thorax at the scutum. The sporozoites were isolated from the separated heads and the anterior portions of the scutum using Ozaki tubes, and then purified using a DEAE column, counted using a hemocytometer and a suspension of 5,000 sporozoites added to wells. Slides were dried overnight and then wrapped in aluminum foil and placed in a desiccator at −20 °C. Polyclonal antibody described above was used for detection of PNAG by immunofluorescence assay (IFA). The control and immune sera were diluted at 1:50 in PBS + 1% BSA, slides thawed for 20 min at room temperature, and 15 µL of each serum sample added to individual wells and incubated for 1 h. The slides were washed three times for 2 min in PBS, then 16 µL of the secondary antibody, goat anti-rabbit IgG (heavy and light chains) conjugated to FITC (Southern Biotech) diluted 1:2,000 in PBS + 1% BSA added to each well. After 1 h of incubation, slides were washed three times for 2 min in PBS. Finally, 1 µL of Vectashield with DAPI (Vector Laboratories) was added to each well and the slide was covered with a coverslip. The slide was covered with aluminum foil until read.

To confirm that sporozoites were expressing PNAG, samples were incubated with 16 µL of chitinase, dispersin B, or sodium periodate as described above for 2 h at 37 °C. Slides were washed three times for 2 min in PBS and then blocked with 16 µL of PBS + 1% BSA. Staining for IFA was then carried out as described in the preceding paragraph. Methods for image analysis of the sporozoite IFA are provided in SI Text 3.

Electron Microscopy.

Details for single and dual antibody labeling techniques and electron microscopic methods are provided in SI Text 3.

In Vitro Opsonic Killing and Bactericidal Assays.

In vitro killing of S. pneumoniae, E. faecalis, S. pyogenes, and C. albicans followed published protocols used with S. aureus (17), except that the differentiated HL60 promyelocytic cell line (ATCC) was used as a source of phagocytes (55). Bactericidal killing of N. gonorrhoeae and N. meningitidis was carried out as described (36) using normal rabbit serum or rabbit immune serum raised to 9GlcNH2-TT (22).

In Vivo Protection Experiments.

Streptococcus pyogenes.

Two groups of 6-wk-old CD1(ICR) female mice were immunized i.p. with 200 µL per mouse of rabbit antibody to 9GlcNH2-TT or normal rabbit sera 24 h and 4 h before infection. GAS strain 950771 grown to midexponential phase in Todd Hewitt broth at 37 °C was pelleted and resuspended to ∼2 × 106 cfu/mL, then 100 µL injected s.c. into the right flank (∼2 × 105 cfu per mouse). Mice were observed twice daily over a 3-d period, and moribund animals killed and counted as dead. Spleens were recovered from the mice, homogenized, and diluted and plated onto blood agar plates to document S. pyogenes dissemination, which was present in all moribund/dead mice.

Listeria monocytogenes and N. meningitidis infections in neonatal mice.

Two-day-old neonatal mice were injected i.p. with 50 µL of either normal or immune serum to 9GlcNH2-TT and 24 h later infected by the i.p. route. For L. monocytogenes-infected mice, survival over 24 h was monitored. Moribund animals were humanely killed and counted as deaths. For N. meningitidis-infected mice, after 24 h the animals were euthanized and brains removed and homogenized, then diluted for quantitative bacterial enumeration.

Streptococcus pneumoniae lung infections.

Inocula of serotype 2 strain D39 were applied to the napes of anesthetized CBA/N mice 24 h after i.p. injection of 200 µL of normal serum or immune serum to PNAG. Survival over the next week was monitored. Moribund animals were humanely killed and counted as deaths.

Inocula of serotype 9V strain DSM 11865 grown at 37 °C with shaking in Mueller–Hinton broth to the end of logarithmic phase was prepared to obtain an inoculum of 9.2 × 109 cfu/mL and shock frozen. For each experiment a fresh vial was thawed and diluted in PBS to obtained 1 × 108 cfu per mouse for intranasal inoculation of FVB mice. The actual inoculums were verified by viable counts. Four hours before infection, mice were treated with a single i.v. dose of mAb F598 or 0.9% NaCl (control group) infected intranasally with 1 × 108 cfu per mouse, and 24 h after infection mice were euthanized by cervical dislocation and lungs were collected for cfu determinations. Cefotaxime was administered at 15 mg/kg by iv route 1 h and 4 h after infection.

Spontaneous and communicable BALB/c T-bet−/− RAG2−/− (TRUC) colitis models and histopathology scoring.

TRUC mice were injected i.p. starting on day 7 of life then every 3 d with either control human IgG1 mAb F105 or anti-PNAG mAb F598 (15 mg/kg) until weaning, then injected on a weekly basis (15 mg/kg). TRUC cross-fostering of BALB/c WT neonatal mice was carried out as described (40, 41). In brief, BALB/c WT pups were provided with a TRUC foster dam within 24 h of birth until weaning at day 21. Pups were injected i.p. starting at 4 wk of age with 15 mg/kg of mAb F105 or mAb F598. At 8 wk of age, colons from all experimental mice were harvested upon sacrifice, and colonic contents removed before fixation in 4% paraformaldehyde. Following paraffin embedding, sections (0.5 μm thick) were cut and stained with hematoxylin and eosin. Histopathology was evaluated in a blinded fashion (with respect to genotype and experimental protocol) by a pathologist using four parameters: mononuclear cell infiltration, polymorphonuclear cell infiltration, epithelial hyperplasia, and epithelial injury that were scored as absent (0), mild (1), moderate (2), or severe (3) as described previously (40, 41).

C. albicans keratitis.

We adapted the mouse model of ulcerative keratitis (56) to evaluate protective efficacy of mAb F598 against fungal infection and associated corneal pathology. C. albicans SC5314 was grown overnight at 30 °C in yeast/peptone/dextrose (YPD) medium, diluted 1:50 into fresh YPD medium and grown for 5 h at 30 °C. Yeast cells were recovered, washed, and resuspended to contain between 105 and 5 × 107 cfu in 5 µL. The corneas of anesthetized mice were scratch injured and 5 µL of the infectious inoculum applied. One group of mice was injected i.p. 24 h before infection with 200 µg of control mAb F429 or mAb F598 to PNAG followed by topical treatment with 5 µg in 5 µL at 24 h and 32 h postinfection. A second group received topical treatment at 4, 8, and 24 h postinfection. Mice were evaluated for corneal pathology on a scale of 1–4 as described (56), killed at 32 or 48 h postinfection, and then eyes processed for enumeration of fungal cells in the cornea. Some eyes were fixed in 4% paraformaldehyde for histopathologic analysis.

P. berghei ANKA infection of mice.

C57BL/6 mice from Jackson Laboratories were bred and maintained under specific pathogen-free conditions in the animal facilities at the University of Massachusetts Medical School. P. berghei ANKA constitutively expressing GFP (GFPcon) was obtained through MR4 as part of the BEI Resources Repository, National Institute of Allergy and Infectious Diseases, National Institutes of Health (P. berghei ANKA GFPcon 259cl2, MRA-865, deposited by C. J. Janse and A. P. Waters) (42). P. berghei ANKA was maintained by passage in C57BL/6 mice. Female C57BL/6 mice, 8–14 wk old, were inoculated i.p. with 1 × 105 infected RBCs. P. berghei ANKA-infected mice succumb to cerebral malaria (CM) between days 7 and 9 postinfection with moderate parasitemia (6–11%). CM was diagnosed by clinical signs, including ruffled fur; unbalanced, deviation of the head; ataxia; paralysis; convulsions; and coma, followed by death (2). Moribund animals were scored as dead and euthanized (57). Mice that did not develop CM were monitored until parasitemia exceeded 50%. Parasitemia was measured using flow cytometry analysis as described elsewhere (58).

Animal experimentation approvals.

All experiments carried out at Harvard Medical School/Brigham and Women’s Hospital were conducted under protocols approved by the Harvard Medical Area Institutional Animal Care and Use Committee. Experiments carried out at the University of Massachusetts Medical School were conducted in accordance with an approved protocol from the Institutional Animal Care and Use Committee. Experiments carried out at Sanofi were approved by the local animal welfare committee, Comité d'Éthique pour la Protection de l'Animal de Laboratoire.

Acknowledgments

We gratefully acknowledge the following individuals for supplying microbial strains or tissue samples: Dr. Peter Rice (University of Massachusetts Medical School); Dr. Lee Wetzler (Boston University Medical School); Drs. Daniel Milner, Eric Rubin, and Marc Lipsitch (Harvard School of Public Health), Drs. Richard Malley and Michael Wessels (Children’s Hospital, Harvard Medical School); Dr. Darren Higgins (Harvard Medical School); Drs. Laurie Comstock, Maria Chatzidaki-Livanis, Neeraj (Neil) Surana, and Lynn Bry (Brigham and Women’s Hospital, Harvard Medical School); Dr. Yung-Fu Chang (Cornell College of Veterinary Medicine); Dr. Joanna Goldberg (University of Virginia Medical School); Drs. Stanley Spinola and Margaret Bauer (Indiana University Medical School); Dr. Darlene Miller (Bascom-Palmer Eye Institute, University of Miami); Dr. Arturo Casadevall (Albert Einstein College of Medicine); Dr. Janet Yother (University of Alabama, Birmingham); Dr. Sandra Richter (University of Iowa); Dr. Janet Lindow (University of Vermont); Dr. Alan Cross (University of Maryland School of Medicine); Dr. Kwang Sik Kim (The Johns Hopkins University School of Medicine); Dr. Rachel McLoughlin (University College Dublin); and Dr. Johannes Huebner (Ludwig Maximilians University). We also gratefully acknowledge the technical expertise and help of Ms. Maria Ericsson with electron microscopy and Ms. Ariana Huebner with laboratory experiments. This work was supported by NIAID Grants AI46706 and AI057159, a component of Award U54 AI057159. The work with T. vaginalis was supported by NIAID Grant 5R01AI079085-04 and the work with P. berghei was supported by NIAID Grant RO1AI079293.

Footnotes

  • ↵1To whom correspondence should be addressed. E-mail: gpier{at}rics.bwh.harvard.edu.
  • Author contributions: C.C.-B., D.S., D.R., R.B.D., W.S.G., X.L., A.R., A.K.K.K., T.M.-L., N.E.N., D.T.G., and G.B.P. designed research; C.C.-B., D.S., Tanweer Zaidi, D.R., R.B.D., W.S.G., X.L., J.O., K.K., Tauqeer Zaidi, A.R., C.P., A.K.K.K., M.L.G., and G.B.P. performed research; W.S.G., R.N.F., T.M.-L., M.L.G., Y.E.T., N.E.N., L.O.B., S.I.P., and D.T.G. contributed new reagents/analytic tools; C.C.-B., D.S., Tanweer Zaidi, D.R., R.B.D., W.S.G., X.L., J.O., K.K., Tauqeer Zaidi, A.R., C.P., R.N.F., A.K.K.K., T.M.-L., M.L.G., Y.E.T., N.E.N., L.O.B., S.I.P., D.T.G., and G.B.P. analyzed data; and C.C.-B., D.S., W.S.G., R.N.F., S.I.P., D.T.G., and G.B.P. wrote the paper.

  • Conflict of interest statement: G.B.P., T.M.-L., M.L.G., Y.E.T., and N.E.N. are inventors of intellectual properties [human monoclonal antibody to PNAG (G.B.P. and T.M.-L.) and PNAG vaccines (G.B.P., T.M.-L., M.L.G., Y.E.T., and N.E.N.], which are licensed by Brigham and Women's Hospital to Alopexx Vaccine, LLC, and Alopexx Pharmaceuticals, LLC, entities in which G.B.P. also holds equity. As inventors of intellectual properties, these authors also have the right to receive a share of licensing-related income (royalties, fees) through Brigham and Women's Hospital from Alopexx Pharmaceuticals, LLC, and Alopexx Vaccine, LLC. G.B.P. and T.M.-L.'s interests were reviewed and are managed by the Brigham and Women's Hospital and Partners Healthcare in accordance with their conflict of interest policies. A.R. and C.P. are employees of Sanofi, Inc., which has licensed the technology for use of mAb F598 (designated as SAR279356) for treatment and prevention of human infections.

  • ↵*This Direct Submission article had a prearranged editor.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1303573110/-/DCSupplemental.

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PNAG as a conserved microbial vaccine target
Colette Cywes-Bentley, David Skurnik, Tanweer Zaidi, Damien Roux, Rosane B. DeOliveira, Wendy S. Garrett, Xi Lu, Jennifer O’Malley, Kathryn Kinzel, Tauqeer Zaidi, Astrid Rey, Christophe Perrin, Raina N. Fichorova, Alexander K. K. Kayatani, Tomas Maira-Litràn, Marina L. Gening, Yury E. Tsvetkov, Nikolay E. Nifantiev, Lauren O. Bakaletz, Stephen I. Pelton, Douglas T. Golenbock, Gerald B. Pier
Proceedings of the National Academy of Sciences Jun 2013, 110 (24) E2209-E2218; DOI: 10.1073/pnas.1303573110

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PNAG as a conserved microbial vaccine target
Colette Cywes-Bentley, David Skurnik, Tanweer Zaidi, Damien Roux, Rosane B. DeOliveira, Wendy S. Garrett, Xi Lu, Jennifer O’Malley, Kathryn Kinzel, Tauqeer Zaidi, Astrid Rey, Christophe Perrin, Raina N. Fichorova, Alexander K. K. Kayatani, Tomas Maira-Litràn, Marina L. Gening, Yury E. Tsvetkov, Nikolay E. Nifantiev, Lauren O. Bakaletz, Stephen I. Pelton, Douglas T. Golenbock, Gerald B. Pier
Proceedings of the National Academy of Sciences Jun 2013, 110 (24) E2209-E2218; DOI: 10.1073/pnas.1303573110
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