Two monoclonal antibodies with defined epitopes of P44 major surface proteins neutralize Anaplasma phagocytophilum by distinct mechanisms.
ABSTRACT: Anaplasma phagocytophilum is an obligatory intracellular bacterium that causes human granulocytic anaplasmosis. The polymorphic 44-kDa major outer membrane proteins of A. phagocytophilum are dominant antigens recognized by patients and infected animals. However, the ability of anti-P44 antibody to neutralize the infection has been unclear due to a mixture of P44 proteins with diverse hypervariable region amino acid sequences expressed by a given bacterial population and lack of epitope-defined antibodies. Monoclonal antibodies (MAbs) 5C11 and 3E65 are directed to different domains of P44 proteins, the N-terminal conserved region and P44-18 central hypervariable region, respectively. Passive immunization with either MAb 5C11 or 3E65 partially protects mice from infection with A. phagocytophilum. In the present study, we demonstrated that the two monoclonal antibodies recognize bacterial surface-exposed epitopes of naturally folded P44 proteins and mapped these epitopes to specific peptide sequences. The two MAbs almost completely blocked the infection of the A. phagocytophilum population that predominantly expressed P44-18 in HL-60 cells by distinct mechanisms: MAb 5C11 blocked the binding, but MAb 3E65 did not block binding or internalization. Instead, MAb 3E65 inhibited internalized A. phagocytophilum to develop into microcolonies called morulae. Some plasma from experimentally infected horses and mice reacted with these two epitopes. Taken together, these data indicate the presence of at least two distinct bacterial surface-exposed neutralization epitopes in P44 proteins. The results indicate that antibodies directed to certain epitopes of P44 proteins have a critical role in inhibiting A. phagocytophilum infection of host cells.
Project description:The 44-kDa immunodominant outer membrane proteins (P44 proteins) of Anaplasma phagocytophilum are encoded by the p44 polymorphic multigene family. The present study examined p44 expression and analyzed the cDNA sequences of various p44 transcripts from the spleens and blood of mice infected by the bites of ticks infected with the A. phagocytophilum NTN-1 strain or of naturally infected nymphal ticks and in the salivary glands and midgut tissues of these ticks. A total of 300 p44 cDNAs were subjected to sequence analysis. Of these, 40 distinct p44 species were found, and all of these had orthologs in the A. phagocytophilum HZ strain genome that shared 95 to 100% base sequence identity. The number of unique p44 species expressed in mouse blood was greater than that for mouse spleens. Higher numbers of different p44 transcripts were also expressed in the salivary glands of ticks than in the midgut tissues. Variations in the sequences of the same p44 cDNA species within a single A. phagocytophilum strain and among different strains were concentrated in the conserved regions flanking the central hypervariable region of p44 genes. No mosaic sequences derived from two or more p44 species were found within the p44 hypervariable region. The conservation of the hypervariable region of each p44 cDNA species of A. phagocytophilum in naturally infected ticks and in different geographic isolates suggests that each A. phagocytophilum genome carries a set of p44 paralogs to be expressed. Thus, a large but restricted repertoire of p44 hypervariable sequences exists in A. phagocytophilum strains in the Northeastern United States.
Project description:The human intragranulocytic bacterium Anaplasma phagocytophilum promotes variation of P44s, which are surface-exposed proteins encoded by a p44 multigene family. In the present study, the specific p44 gene expression loci in four strains of A. phagocytophilum were identified and it was determined that each consisted of four tandem genes, tr1, omp-1X, omp-1N, and p44. A putative sigma(70)-type promoter was found upstream of tr1. The p44 genes include a central hypervariable region flanked by conserved regions. The hypervariable region sequence in the p44 expression locus was duplicated and, regardless of the expression status, conserved at another locus in both low- and high-passage cell cultures of strain NY-37. No significant differences in the hypervariable region were found when we compared p44 sequences, at the level of cDNA, within the expression locus and within other loci in the genomes of strains NY-37 and HZ. Similarly, in cDNA isolated from patients and from assorted cultures of strains NY-31, NY-36, and NY-37, hypervariable regions of 450 deduced amino acid sequences of various p44s within each strain were found to be identical, as were those of p44 sequences in the genome of strain HZ. These data suggest that variations in p44 sequences at the level of the p44 expression locus occur through unidirectional conversion of the entire (nonsegmental) p44 hypervariable region including flanking regions with a corresponding sequence copied from one of the conserved donor p44 genomic loci. The data suggest that the P44 antigenic repertoire within the hypervariable region is restricted.
Project description:Anaplasma phagocytophilum is the causative agent of tick-borne fever in small ruminants and has been identified as the zoonotic agent of human granulocytic anaplasmosis. The Norwegian strains of the rickettsia are naturally persistent in lambs and represent a suitable experimental system for analyzing the mechanisms of persistence. Variation of the outer membrane protein MSP2(P44) by recombination of variable pseudogene segments into an expression site is believed to play a key role in persistence of the organism. The goal of the present study was to analyze the dynamics of the immune response towards A. phagocytophilum and MSP2(P44) during persistent infection of lambs. Responses to the hypervariable region of MSP2(P44) were detected shortly after appearance of the respective variants in cyclic rickettsemic peaks, consistent with a process of antigenic variation. In addition, there was a diminishing antibody response to MSP2(P44) and to other A. phagocytophilum antigens overall with time of infection, that was not associated with clearance of the infection.
Project description:Carbohydrate antigens are important targets of the immune system in clearing bacterial pathogens. Although the immune system almost exclusively uses antibodies in response to foreign carbohydrates, there is still much to learn about the role of different epitopes on the carbohydrate as targets of protective immunity. We examined the role of acetyl group-dependent and -independent epitopes on the staphylococcal surface of polysaccharide poly-N-acetylated glucosamine (PNAG) by use of human monoclonal antibodies (MAbs) specific for such epitopes. We utilized hybridoma technology to produce fully human immunoglobulin G2 (IgG2) MAbs from B cells of an individual post-Staphylococcus aureus infection and cloned the antibody variable regions to produce an IgG1 form of each original MAb. Specificity and functionality of the purified MAbs were tested in vitro using enzyme-linked immunosorbent assays, complement deposition, and opsonophagocytic assays. We found that a MAb (MAb F598) that bound the best to nonacetylated or backbone epitopes on PNAG had superior complement deposition and opsonophagocytic activity compared to two MAbs that bound optimally to PNAG that was expressed with a native level (>90%) of N-acetyl groups (MAbs F628 and F630). Protection of mice against lethality due to S. aureus strains Mn8 and Reynolds further showed that the backbone-specific MAb had optimal protective efficacy compared with the acetate-specific MAbs. These results provide evidence for the importance of epitope specificity in inducing the optimal protective antibody response to PNAG and indicate that MAbs to the deacetylated form of PNAG could be immunotherapeutic agents for preventing or treating staphylococcal infections.
Project description:Bacterial biofilms are recalcitrant to antibiotic therapy and a major cause of persistent and recurrent infections. New antibody-based therapies may offer potential to target biofilm specific components for host-cell mediated bacterial clearance. For Pseudomonas aeruginosa, human monoclonal antibodies (mAbs) targeting the Psl biofilm exopolysaccharide exhibit protective activity against planktonic bacteria in acute infection models. However, anti-Psl mAb activity against P. aeruginosa biofilms is unknown. Here, we demonstrate that anti-Psl mAbs targeting three distinct Psl epitopes exhibit stratified binding in mature in vitro biofilms and bind Psl within the context of a chronic biofilm infection. These mAbs also exhibit differential abilities to inhibit early biofilm events and reduce biomass from mature biofilms in the presence of neutrophils. Importantly, a mAb mixture with neutrophils exhibited the greatest biomass reduction, which was further enhanced when combined with meropenem, a common anti-Pseudomonal carbapenem antibiotic. Moreover, neutrophil-mediated killing of biofilm bacteria correlated with the evident mAb epitope stratification within the biofilm. Overall, our results suggest that anti-Psl mAbs might be promising candidates for adjunctive use with antibiotics to inhibit/disrupt P. aeruginosa biofilms as a result of chronic infection.
Project description:Characterization of the epitopes on antigen recognized by monoclonal antibodies (mAb) is useful for the development of therapeutic antibodies, diagnostic tools, and vaccines. Epitope mapping also provides functional information for sequence-based repertoire analysis of antibody response to pathogen infection and/or vaccination. However, development of mapping strategies has lagged behind mAb discovery. We have developed a site-directed mutagenesis approach that can be used in conjunction with bio-layer interferometry (BLI) biosensors to map mAb epitopes. By generating a panel of single point mutants in the recombinant hemagglutinin (HA) and neuraminidase (NA) proteins of influenza A viruses, we have characterized the epitopes of hundreds of mAbs targeting the H1 and H3 subtypes of HA and the N9 subtype of NA.
Project description:The natural life cycle of Anaplasma phagocytophilum, an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, consists of alternate infection of two distinct hosts, ticks and mammals, in which bacterial surface proteins are expected to have a critical role. The present study investigated regulation of A. phagocytophilum p44 genes, which encode the P44 major surface proteins. Quantitative real-time reverse transcription-PCR analysis revealed that the amount of p44 mRNA obtained from spleens of A. phagocytophilum-infected SCID mice was approximately 10-fold greater than the amount obtained from salivary glands of A. phagocytophilum-infected Ixodes scapularis nymphs. Similarly, the amount of p44 mRNA obtained from A. phagocytophilum-infected HL-60 cells per bacterium was significantly greater than the amount obtained from infected ISE6 tick cells. The relative amount of p44 mRNA was approximately threefold higher in A. phagocytophilum-infected HL-60 cells cultured at 37 degrees C than in A. phagocytophilum-infected HL-60 cells cultured at 28 degrees C. Although there are more than 100 p44 paralogs, we observed expression mainly from the p44 expression locus (p44E) in various host environments. Interestingly, transcription of the A. phagocytophilum gene encoding the DNA binding protein ApxR was also significantly greater in A. phagocytophilum-infected HL-60 cells than in infected ISE6 tick cells. Gel mobility shift and DNase I protection assays revealed recombinant ApxR binding to the promoter regions of p44E and apxR. ApxR also transactivated the p44E and apxR promoter regions in a lacZ reporter assay. These results indicate that p44 genes and apxR are specifically up-regulated in the mammalian host environment and suggest that ApxR not only is positively autoregulated but also acts as a transcriptional regulator of p44E.
Project description:BACKGROUND:Severe acute respiratory syndrome (SARS) remains a significant public health concern after the epidemic in 2003. Human monoclonal antibodies (MAbs) that neutralize SARS-associated coronavirus (SARS-CoV) could provide protection for exposed individuals. METHODS:Transgenic mice with human immunoglobulin genes were immunized with the recombinant major surface (S) glycoprotein ectodomain of SARS-CoV. Epitopes of 2 neutralizing MAbs derived from these mice were mapped and evaluated in a murine model of SARS-CoV infection. RESULTS:Both MAbs bound to S glycoprotein expressed on transfected cells but differed in their ability to block binding of S glycoprotein to Vero E6 cells. Immunoprecipitation analysis revealed 2 antibody-binding epitopes: one MAb (201) bound within the receptor-binding domain at aa 490-510, and the other MAb (68) bound externally to the domain at aa 130-150. Mice that received 40 mg/kg of either MAb prior to challenge with SARS-CoV were completely protected from virus replication in the lungs, and doses as low as 1.6 mg/kg offered significant protection. CONCLUSIONS:Two neutralizing epitopes were defined for MAbs to SARS-CoV S glycoprotein. Antibodies to both epitopes protected mice against SARS-CoV challenge. Clinical trials are planned to test MAb 201, a fully human MAb specific for the epitope within the receptor-binding region.
Project description:Diverse p44 alleles at the p44 expression locus (p44Es) encoding surface-exposed major membrane proteins, P44s, of Anaplasma phagocytophilum were hypothesized to be garnered by recombination to enact antigenic variation. However, this hypothesis has not been proven so far, due to inability to clone this obligate intragranulocytic rickettsia. To define the p44E recombination, we developed a novel method to clone A. phagocytophilum. This isogenic cloned population containing a defined p44E was used to infect a naive horse and severe combined immunodeficiency (SCID) mice. During a 58-day infection period in the blood of the horse, p44E conversion was evident in a total of 11 new p44Es, 48% (115/242) of the sequenced p44E population. During a 50-day infection period in the blood of SCID mice, p44E conversion was manifested in a total of 13 new p44Es, 42% (192/460) of the p44E population. Thus, similar levels of p44E convertants were detected in either the presence or absence of an acquired immune system, suggesting that T- and B-cell immune pressure was not essential for recombination and/or selection of the p44E variants. Analysis of sequentially changed p44Es revealed that the entire central hypervariable region of donor p44 pseudogenes or of donor full-length p44s replaced the same region of the resident p44E as a cassette. Putative recombination points were detected within p44 conserved regions flanking the central hypervariable region by the TOPALi analysis. Our results unambiguously demonstrated p44E recombination. The cloning method developed would facilitate precise analysis of the recombination process and the extent of diversity which the recombination creates in the antigenic repertoire.
Project description:Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 microgram/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 micrograms/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.