Solomon Langermann

Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences.

Many biological functions, including control of the homeostasis and maternofetal transfer of serum γ-globulins, are mediated by the MHC class I-related neonatal FcR (FcRn). A correlation exists in mice between the binding affinity of IgG1/Fc fragments to FcRn at pH 6.0 and their serum t1/2. To expand this observation, phage display of mutagenized Fc fragments derived from a human IgG1 was used to increase their affinity to both murine and human FcRn. Ten variants were identified that have a higher affinity toward murine and human FcRn at pH 6.0, with ΔΔG (ΔGwild type − ΔGmutant) from 1.0 to 2.0 kcal/mol and from 0.6 to 2.4 kcal/mol, respectively. Those variants exhibit a parallel increase in binding at pH 7.4 to murine, but not human, FcRn. Although not degraded in blood in vitro, accumulated in tissues, nor excreted in urine, their serum concentration in mice is decreased. We propose that higher affinity to FcRn at pH 7.4 adversely affects release into the serum and offsets the benefit of the enhanced binding at pH 6.0.

Use of a Whole Genome Approach To Identify Vaccine Molecules Affording Protection against Streptococcus pneumoniae Infection

ABSTRACT Microbial targets for protective humoral immunity are typically surface-localized proteins and contain common sequence motifs related to their secretion or surface binding. Exploiting the whole genome sequence of the human bacterial pathogen Streptococcus pneumoniae, we identified 130 open reading frames encoding proteins with secretion motifs or similarity to predicted virulence factors. Mice were immunized with 108 of these proteins, and 6 conferred protection against disseminated S. pneumoniaeinfection. Flow cytometry confirmed the surface localization of several of these targets. Each of the six protective antigens showed broad strain distribution and immunogenicity during human infection. Our results validate the use of a genomic approach for the identification of novel microbial targets that elicit a protective immune response. These new antigens may play a role in the development of improved vaccines against S. pneumoniae.

Structural basis of tropism of Escherichia coli to the bladder during urinary tract infection

The first step in the colonization of the human urinary tract by pathogenic Escherichia coli is the mannose‐sensitive binding of FimH, the adhesin present at the tip of type 1 pili, to the bladder epithelium. We elucidated crystallographically the interactions of FimH with D‐mannose. The unique site binding pocket occupied by D‐mannose was probed using site‐directed mutagenesis. All but one of the mutants examined had greatly diminished mannose‐binding activity and had also lost the ability to bind human bladder cells. The binding activity of the mono‐saccharide D‐mannose was delineated from this of mannotriose (Man(α1–3)[Man(α1–6)]Man) by gener‐ating mutants that abolished D‐mannose binding but retained mannotriose binding activity. Our structure/function analysis demonstrated that the binding of the monosaccharide α‐D‐mannose is the primary bladder cell receptor for uropathogenic E. coli and that this event requires a highly conserved FimH binding pocket. The residues in the FimH mannose‐binding pocket were sequenced and found to be invariant in over 200 uropathogenic strains of E. coli. Only enterohaemorrhagic E. coli (EHEC) possess a sequence variation within the mannose‐binding pocket of FimH, suggesting a naturally occurring mechanism of attenuation in EHEC bacteria that would prevent them from being targeted to the urinary tract.

Receptor binding studies disclose a novel class of high‐affinity inhibitors of the Escherichia coli FimH adhesin

Mannose‐binding type 1 pili are important virulence factors for the establishment of Escherichia coli urinary tract infections (UTIs). These infections are initiated by adhesion of uropathogenic E. coli to uroplakin receptors in the uroepithelium via the FimH adhesin located at the tips of type 1 pili. Blocking of bacterial adhesion is able to prevent infection. Here, we provide for the first time binding data of the molecular events underlying type 1 fimbrial adherence, by crystallographic analyses of the FimH receptor binding domains from a uropathogenic and a K‐12 strain, and affinity measurements with mannose, common mono‐ and disaccharides, and a series of alkyl and aryl mannosides. Our results illustrate that the lectin domain of the FimH adhesin is a stable and functional entity and that an exogenous butyl α‐ d‐mannoside, bound in  the  crystal  structures,  exhibits  a  significantly better affinity for FimH (Kd = 0.15 µM) than mannose (Kd = 2.3 µM). Exploration of the binding affinities of α‐ d‐mannosides with longer alkyl tails revealed affinities up to 5 nM. Aryl mannosides and fructose can also bind with high affinities to the FimH lectin domain, with a 100‐fold improvement and 15‐fold reduction in affinity, respectively, compared with mannose. Taken together, these relative FimH affinities correlate exceptionally well with the relative concentrations of the same glycans needed for the inhibition of adherence of type 1 piliated E. coli. We foresee that our findings will spark new ideas and initiatives for the development of UTI vaccines and anti‐adhesive drugs to prevent anticipated and recurrent UTIs.

Vaccination with FimH Adhesin Protects Cynomolgus Monkeys from Colonization and Infection by Uropathogenic Eschevichia coli

Escherichia coli FimH adhesin mediates binding to the bladder mucosa. In mice, a FimH vaccine protects against bacterial challenge. In this study, 4 monkeys were inoculated with 100 microgram of FimCH adhesin-chaperone complex mixed with MF59 adjuvant, and 4 monkeys were given adjuvant only intramuscularly. After 2 doses (day 0 and week 4), a booster at 48 weeks elicited a strong IgG antibody response to FimH in the vaccinated monkeys. All 8 monkeys were challenged with 1 mL of 108 E. coli cystitis isolate NU14. Three of the 4 vaccinated monkeys were protected from bacteruria and pyuria; all control monkeys were infected. These findings suggest that a vaccine based on the FimH adhesin of E. coli type 1 pili may have utility in preventing cystitis in humans.

Identification and Characterization of a Novel Family of Pneumococcal Proteins That Are Protective against Sepsis

ABSTRACT Four pneumococcal genes (phtA, phtB, phtD, andphtE) encoding a novel family of homologous proteins (32 to 87% identity) were identified from the Streptococcus pneumoniae genomic sequence. These open reading frames were selected as potential vaccine candidates based upon their possession of hydrophobic leader sequences which presumably target these proteins to the bacterial cell surface. Analysis of the deduced amino acid sequences of these gene products revealed the presence of a histidine triad motif (HxxHxH), termed Pht (pneumococcal histidine triad) that is conserved and repeated several times in each of the four proteins. The four pht genes (phtA, phtB, phtD, and a truncated version of phtE) were expressed inEscherichia coli. A flow cytometry-based assay confirmed that PhtA, PhtB, PhtD and, to a lesser extent, PhtE were detectable on the surface of intact bacteria. Recombinant PhtA, PhtB, and PhtD elicited protection against certain pneumococcal capsular types in a mouse model of systemic disease. These novel pneumococcal antigens may serve as effective vaccines against the most prevalent pneumococcal serotypes.

Adhesins as targets for vaccine development.

Blocking the primary stages of infection, namely bacterial attachment to host cell receptors and colonization of the mucosal surface, may be the most effective strategy to prevent bacterial infections. Bacterial attachment usually involves an interaction between a bacterial surface protein called an adhesin and the host cell receptor. Recent preclinical vaccine studies with the FimH adhesin (derived from uropathogenic Escherichia coli) have confirmed that antibodies elicited against an adhesin can impede colonization, block infection, and prevent disease. The studies indicate that prophylactic vaccination with adhesins can block bacterial infections. With recent advances in the identification, characterization, and isolation of other adhesins, similar approaches are being explored to prevent infections, from otitis media and dental caries to pneumonia and sepsis.

Vaccination Utilizing the FimCH Complex As a Strategy to Prevent Escherichia coli Urinary Tract Infections

Eseherichia coli are the main causative agents of uncomplicated urinary tract infection (UTI) and account for >85% of cases of acute cystitis and pyelonephritis, >60% of recurrent cystitis, and at least 35% of recurrent pyelonephritis [1]. The reservoir for uropathogenic E. coli is fecal flora, from which bacteria spread to the urogenital mucosa, ascend into the bladder, and adhere to bladder epithelium. Once established in the bladder, the bacteria multiply and establish a local infection (cystitis) and may further ascend to involve the ureters and kidneys (pyelonephritis). While many factors contribute to the acquisition and progression of E. coli UTI, colonization of the urogenital tract by pathogenic bacteria appears to be a prerequisite for disease [2]. One promising strategy for the prevention of UTI is the development of a vaccine that targets the mechanism(s) by which uropathogens adhere to bladder epithelial cells.

PC Cell-Derived Growth Factor Expression in Prostatic Intraepithelial Neoplasia and Prostatic Adenocarcinoma

Purpose: PCDGF (PC cell-derived growth factor), also called progranulin, is a Mr 88,000 glycoprotein precursor of granulin. It is a novel growth factor that stimulates cell proliferation, confers epithelial tumorigenesis, and promotes tumor invasion. Here we investigate the potential of PCDGF as a therapeutic target for prostate cancer. Experimental Design: We studied the expression of PCDGF in invasive prostate cancer, adjacent high-grade prostatic intraepithelial neoplasia (PIN), and benign prostate tissue from 99 human prostate specimens. The level of PCDGF expression was correlated with various clinicopathological characteristics. Results: Normal prostate tissue did not express (53/99), or expressed low levels (46/99) of PCDGF. In the 46 normal prostate specimens that expressed PCDGF, most of them had less than 10% of cells expressing PCDGF. PCDGF expression could be detected in more than 50% of cells in all specimens of PIN and invasive prostate cancer. The expression of PCDGF in normal prostate tissue was much less intense and in a smaller fraction of cells than in PIN and invasive adenocarcinoma (P < 0.0001). There was no correlation of PCDGF expression with age, Gleason score, pathological stage, status of lymph node metastasis, extraprostatic extension, perineural invasion, surgical margins, and vascular invasion. Conclusions: Our data suggest that the induction of PCDGF expression occurs during the development of PIN. PCDGF may be a new molecular target for the treatment and prevention of prostate cancer.

B7-H4Ig inhibits mouse and human T-cell function and treats EAE via IL-10/Treg-dependent mechanisms

We evaluated the therapeutic efficacy and mechanisms of action of both mouse and human B7-H4 Immunoglobulin fusion proteins (mB7-H4Ig; hB7-H4Ig) in treating EAE. The present data show that mB7-H4Ig both directly and indirectly (via increasing Treg function) inhibited CD4⁺ T-cell proliferation and differentiation in both Th1- and Th17-cell promoting conditions while inducing production of IL-10. B7-H4Ig treatment effectively ameliorated progression of both relapsing (R-EAE) and chronic EAE correlating with decreased numbers of activated CD4⁺ T-cells within the CNS and spleen, and a concurrent increase in number and function of Tregs. The functional requirement for Treg activation in treating EAE was demonstrated by a loss of therapeutic efficacy of hB7-H4Ig in R-EAE following inactivation of Treg function either by anti-CD25 treatment or blockade of IL-10. Significant to the eventual translation of this treatment into clinical practice, hB7-H4Ig similarly inhibited the in vitro differentiation of naïve human CD4⁺ T-cells in both Th1- and Th17-promoting conditions, while promoting the production of IL-10. B7-H4Ig thus regulates pro-inflammatory T-cell responses by a unique dual mechanism of action and demonstrates significant promise as a therapeutic for autoimmune diseases, including MS.