Human monoclonal antibodies inhibit invasion of transgenic Plasmodium knowlesi expressing Plasmodium vivax Duffy binding protein.

BACKGROUND: Plasmodium vivax has been more resistant to various control measures than Plasmodium falciparum malaria because of its greater transmissibility and ability to produce latent parasite forms. Therefore, developing P. vivax vaccines and therapeutic monoclonal antibodies (humAbs) remains a high priority. The Duffy antigen receptor for chemokines (DARC) expressed on erythrocytes is central to P. vivax invasion of reticulocytes. P. vivax expresses a Duffy binding protein (PvDBP) on merozoites, a DARC ligand, and the DARC: PvDBP interaction is critical for P. vivax blood stage malaria. Therefore, PvDBP is a leading vaccine candidate for P. vivax and a target for therapeutic human monoclonal antibodies (humAbs). METHODS: Here, the functional activity of humAbs derived from naturally exposed and vaccinated individuals are compared for the first time using easily cultured Plasmodium knowlesi (P. knowlesi) that had been genetically modified to replace its endogenous PkDBP orthologue with PvDBP to create a transgenic parasite, PkPvDBPOR. This transgenic parasite requires DARC to invade human erythrocytes but is not reticulocyte restricted. This model was used to evaluate the invasion inhibition potential of 12 humAbs (9 naturally acquired; 3 vaccine-induced) targeting PvDBP individually and in combinations using growth inhibition assays (GIAs). RESULTS: The PvDBP-specific humAbs demonstrated 70-100% inhibition of PkPvDBPOR invasion with the IC50 values ranging from 51 to 338 µg/mL for the 9 naturally acquired (NA) humAbs and 33 to 99 µg/ml for the 3 vaccine-induced (VI) humAbs. To evaluate antagonistic, additive, or synergistic effects, six pairwise combinations were performed using select humAbs. Of these combinations tested, one NA/NA (099100/094083) combination demonstrated relatively strong additive inhibition between 10 and 100 µg/mL; all combinations of NA and VI humAbs showed additive inhibition at concentrations below 25 µg/mL and antagonism at higher concentrations. None of the humAb combinations showed synergy. Invasion inhibition efficacy by some mAbs shown with PkPvDBPOR was closely replicated using P. vivax clinical isolates. CONCLUSION: The PkPvDBPOR transgenic model is a robust surrogate of P. vivax to assess invasion and growth inhibition of human monoclonal Abs recognizing PvDBP individually and in combination. There was no synergistic interaction for growth inhibition with the humAbs tested here that target different epitopes or subdomains of PvDBP, suggesting little benefit in clinical trials using combinations of these humAbs.

Identification and Characterization of Functional Human Monoclonal Antibodies to Plasmodium vivax Duffy-Binding Protein.

Plasmodium vivax invasion of reticulocytes relies on distinct receptor-ligand interactions between the parasite and host erythrocytes. Engagement of the highly polymorphic domain II of the P. vivax Duffy-binding protein (DBPII) with the erythrocyte's Duffy Ag receptor for chemokines (DARC) is essential. Some P. vivax-exposed individuals acquired Abs to DBPII that block DBPII-DARC interaction and inhibit P. vivax reticulocyte invasion, and Ab levels correlate with protection against P. vivax malaria. To better understand the functional characteristics and fine specificity of protective human Abs to DBPII, we sorted single DBPII-specific IgG+ memory B cells from three individuals with high blocking activity to DBPII. We identified 12 DBPII-specific human mAbs from distinct lineages that blocked DBPII-DARC binding. All mAbs were P. vivax strain transcending and targeted known binding motifs of DBPII with DARC. Eleven mAbs competed with each other for binding, indicating recognition of the same or overlapping epitopes. Naturally acquired blocking Abs to DBPII from individuals with high levels residing in different P. vivax-endemic areas worldwide competed with mAbs, suggesting broadly shared recognition sites. We also found that mAbs inhibited P. vivax entry into reticulocytes in vitro. These findings suggest that IgG+ memory B cell activity in individuals with P. vivax strain-transcending Abs to DBPII display a limited clonal response with inhibitory blocking directed against a distinct region of the molecule.

Potent AMA1-specific human monoclonal antibody against P. vivax Pre-erythrocytic and Blood Stages.

New therapeutics are a priority for preventing and eliminating Plasmodium vivax (Pv) malaria because of its easy transmissibility and dormant stages in the liver. Relapses due to the dormant liver stages are the major contributor to reoccurring Pv. Therefore, therapies that reduce the establishment of dormant parasites and blood-stage infection are important for controlling this geographically widespread parasite. Here, we isolated 12 human monoclonal antibodies (humAbs) from the plasma of a Pv-exposed individual that recognized Pv apical membrane antigen 1 (PvAMA1). PvAMA1 is important for both sporozoite invasion of hepatocytes and merozoite invasion of reticulocytes. We identified one humAb, 826827, that blocked invasion of human erythrocytes using a transgenic P. falciparum line expressing PvAMA1 (IC 50 = 3 µg/mL) and all Pv clinical isolates in vitro . This humAb also inhibited sporozoite invasion of a human hepatocyte cell line and primary human hepatocytes (IC 50 of 0.3 - 3.7 µg/mL). The crystal structure of recombinant PvAMA1 with the antigen-binding fragment of 826827 at 2.4 Å resolution shows that the humAb partially occupies the highly conserved hydrophobic groove in PvAMA1 that binds its known receptor, RON2. HumAb 826827 binds to PvAMA1 with higher affinity than RON2, accounting for its potency. To our knowledge, this is the first reported humAb specific to PvAMA1, and the PvAMA1 residues it binds to are highly conserved across different isolates, explaining its strain-transcendent properties.

Naturally-acquired and Vaccine-induced Human Monoclonal Antibodies to Plasmodium vivax Duffy Binding Protein Inhibit Invasion of Plasmodium knowlesi (PvDBPOR) Transgenic Parasites.

The Duffy antigen receptor for chemokines (DARC) expressed on erythrocytes is central to Plasmodium vivax (Pv) invasion of reticulocytes. Pv expresses a Duffy binding protein (PvDBP) on merozoites, a DARC ligand, and their protein-protein interaction is central to vivax blood stage malaria. Here we compared the functional activity of humAbs derived from naturally exposed and vaccinated individuals for the first time using easily cultured P. knowlesi (Pk) that had been genetically modified to replace its endogenous PkDBP orthologue with PvDBP to create a transgenic parasite, PkPvDBPOR. This transgenic parasite requires DARC to invade human erythrocytes but is not reticulocyte restricted. Using this model, we evaluated the invasion inhibition potential of 12 humAbs (9 naturally acquired; 3 vaccine-induced) targeting PvDBP individually and in combinations using growth inhibition assays (GIAs). The PvDBP-specific humAbs demonstrated 70-100% inhibition of PkPvDBPOR invasion with the IC50 values ranging from 51 to 338 µg/mL for the 9 naturally acquired (NA) humAbs and 33 to 99 µg/ml for the 3 vaccine-induced (VI) humAbs. To evaluate antagonistic, additive, or synergistic effects, six pairwise combinations were performed using select humAbs. Of these combinations tested, one NA/NA (099100/094083) combination demonstrated relatively strong additive inhibition between 10-100 µg/mL; all combinations of NA and VI humAbs showed additive inhibition at concentrations below 25 µg/mL and antagonism at higher concentrations. None of the humAb combinations showed synergy. This PkPvDBPOR model system enables efficient assessment of NA and VI humAbs individually and in combination.

Naturally acquired blocking human monoclonal antibodies to Plasmodium vivax reticulocyte binding protein 2b.

Plasmodium vivax preferentially invades reticulocytes and recognition of these cells is mediated by P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) binding to human Transferrin receptor 1 (TfR1) and Transferrin (Tf). Longitudinal cohort studies in Papua New Guinea, Thailand and Brazil show that PvRBP2b antibodies are correlated with protection against P. vivax infection and disease. Here, we isolate and characterize anti-PvRBP2b human monoclonal antibodies from two individuals in Cambodia with natural P. vivax infection. These antibodies bind with high affinities and map to different regions of PvRBP2b. Several human antibodies block PvRBP2b binding to reticulocytes and inhibit complex formation with human TfR1-Tf. We describe different structural mechanisms for functional inhibition, including either steric hindrance with TfR1-Tf or the reticulocyte membrane. These results show that naturally acquired human antibodies against PvRBP2b can inhibit its function which is important for P. vivax invasion.

Multiple copies of functional, Tet(M)-encoding Tn916-like elements in a clinical Enterococcus faecium isolate.

Tn916 and similar elements are very common in clinical enterococcal isolates, and are responsible for transmission of a variety of resistance determinants. It is commonly assumed that clinical strains carrying Tn916 have a single copy, although the actual number of copies in clinical isolates has never been systematically studied. We report a clinical isolate of Enterococcus faecium in which three distinct and excision-proficient copies of Tn916-like elements are present in the genome. All of the elements contain tet(M) genes, at least one of which confers resistance to tetracycline and minocycline. Two elements (Tn6085a, Tn6085b) are indistinguishable, containing an inserted 2758bp Group II intron at the start of open reading frame Tn916ORF_06. The third (Tn6084) also contains the intron, but also has an ISEfa11 integrated upstream of tet(M). All three copies are able to excise from plasmid vectors when cloned in E. coli, and at least two of the elements can transfer to an E. faecium recipient strain. These data indicate that nearly identical Tn916-like elements encoding Tet(M)-mediated tetracycline/minocycline resistance can coexist in clinical E. faecium isolates.

Amplification of Duffy binding protein-encoding gene allows Plasmodium vivax to evade host anti-DBP humoral immunity.

Antigenic variation, the capacity to produce a range of variable antigens, is a well-described strategy of Plasmodium and other parasites to evade host immunity. Here, we show that gene amplification is an additional evasion mechanism used by Plasmodium vivax to escape humoral immunity targeting PvDBP, the key ligand involved in reticulocyte invasion. PvDBP gene amplification leads to increased mRNA levels and protects P. vivax in vitro against invasion inhibitory human monoclonal antibodies targeting a conserved binding domain of DBP. Patient samples suggest that parasites with increased pvdbp copy number are able to infect individuals with naturally acquired antibodies highly blocking the binding of PvDBP to the Duffy receptor. These results show that gene copy number variation affect the parasite's ability to evade anti-PvDBP humoral immunity.

Role of class A penicillin-binding proteins in the expression of beta-lactam resistance in Enterococcus faecium.

Peptidoglycan is polymerized by monofunctional d,d-transpeptidases belonging to class B penicillin-binding proteins (PBPs) and monofunctional glycosyltransferases and by bifunctional enzymes that combine both activities (class A PBPs). Three genes encoding putative class A PBPs (pbpF, pbpZ, and ponA) were deleted from the chromosome of Enterococcus faecium D344R in all possible combinations in order to identify the glycosyltransferases that cooperate with low-affinity class B Pbp5 for synthesis of peptidoglycan in the presence of beta-lactam antibiotics. The viability of the triple mutant indicated that glycan strands can be polymerized independently from class A PBPs by an unknown glycosyltranferase. The susceptibility of the DeltapbpF DeltaponA mutant and triple mutants to extended spectrum cephalosporins (ceftriaxone and cefepime) identified either PbpF or PonA as essential partners of Pbp5 for peptidoglycan polymerization in the presence of the drugs. Mass spectrometry analysis of peptidoglycan structure showed that loss of PonA and PbpF activity led to a minor decrease in the extent of peptidoglycan cross-linking by the remaining PBPs without any detectable compensatory increase in the participation of the L,D-transpeptidase in peptidoglycan synthesis. Optical density measurements and electron microscopy analyses showed that the DeltapbpF DeltaponA mutant underwent increased stationary-phase autolysis compared to the parental strain. Unexpectedly, deletion of the class A pbp genes revealed dissociation between the expression of resistance to cephalosporins and penicillins, although the production of Pbp5 was required for resistance to both classes of drugs. Thus, susceptibility of Pbp5-mediated peptidoglycan cross-linking to different beta-lactam antibiotics differed as a function of its partner glycosyltransferase.

The KQ element, a complex genetic region conferring transferable resistance to carbapenems, aminoglycosides, and fluoroquinolones in Klebsiella pneumoniae.

The bla(KPC-3) and qnrB19 determinants of transferable Klebsiella pneumoniae plasmid pLRM24 reside within a complex region consisting of a Tn1331 backbone into which a Tn4401-like element and qnrB19 mobilized by an adjacent ISEcp1 insertion sequence have been inserted. This novel element represents a coalescence of genes conferring multidrug resistance in K. pneumoniae.

Presence of plasmid-mediated quinolone resistance in Klebsiella pneumoniae isolates possessing blaKPC in the United States.

The presence of plasmid-mediated quinolone resistance genes [i.e., qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA] was evaluated among 42 bla(KPC)-containing Klebsiella pneumoniae isolates collected in the eastern United States. One isolate carried the bla(KPC-3) and qnrB19 genes on the same conjugative plasmid, whereas another carried the bla(KPC-3) and qnrA1 genes on separate plasmids.

Transferable capacity for gastrointestinal colonization in Enterococcus faecium in a mouse model.

A high level of gastrointestinal colonization frequently precedes invasive infection due to Enterococcus faecium. Factors other than antimicrobial resistance that promote gastrointestinal colonization by E. faecium have not been identified. We tested the ability of a colonization-proficient clinical E. faecium isolate (C68) to transfer colonizing ability to noncolonizing E. faecium recipient strains. Transconjugants derived from matings that used E. faecium D344SRF as a recipient strain colonized mouse gastrointestinal tracts in high numbers under selective pressure from clindamycin or vancomycin, compared with control strains that lacked DNA transferred from C68. We transferred DNA into a second recipient strain (E. faecium GE-1), which also colonized mice in significantly greater numbers under selective pressure from clindamycin, compared with a control strain. These results indicate that E. faecium clinical isolates express transmissible factors other than antimicrobial resistance that promote colonization of the mouse gastrointestinal tract.

Interaction of related Tn916-like transposons: analysis of excision events promoted by Tn916 and Tn5386 integrases.

In recent work, we described the excision of a large genomic region from Enterococcus faecium D344R in which the sequence from "joint" regions suggested that excision resulted from the interaction of conjugative transposon Tn916 and the related mobile element Tn5386. In the present study, we examined the ability of integrases and integrase-excisase combinations from Tn916 and Tn5386 to promote the excision of constructs consisting of the termini of Tn916, Tn5386, and the VanB mobile element Tn5382. Integrases alone from either Tn916 or Tn5386 promoted the circularization of constructs from the three different transposons, even when the different termini used in the constructs were discordant in their transposon of origin. The termini of Tn916 and Tn5382 found in all joints were consistent with previously identified Tn916 and Tn5382 termini. Substantial variation was seen in the integrase terminus of Tn5386 used to form joints, regardless of the integrase that was responsible for circularization. Variability was observed in joints formed from Tn5386 constructs, in contrast to joints observed with the termini of Tn916 or Tn5382. The coexpression of excisase yielded some variability in the joint regions observed. These data confirm that integrases from some Tn916-like elements can promote circularization with termini derived from heterologous transposons and, as such, could promote excision of large genomic regions flanked by such elements. These findings also raise interesting questions about the sequence specificities of the C terminals of Tn916-like integrases, which bind to the ends and facilitate strand exchange.

Characterization of Tn5386, a Tn916-related mobile element.

In recent work, we described excision of a large genomic region from Enterococcus faecium D344R resulting from the interaction of Tn916 and a related transposon designated Tn5386. In the present study, we present and analyze the complete sequence of Tn5386. Tn5386 is 29,451 bp in length. Fifteen of its 30 open reading frames are analogous to ORFs found in Tn916. Significant differences include a series of ORFs with homology to lantibiotic immunity genes in the same location where tetM is found in Tn916, insertion of a Group II intron and an ORF with similarities to previously described surface exposed collagen adhesion proteins. Our results indicate that Tn5386 falls within the Tn916 family of transposons, and in place of tetM encodes a novel region that may confer resistance to lantibiotics.

Enterococcus faecium low-affinity pbp5 is a transferable determinant.

Using 15 unrelated Enterococcus faecium isolates as donors, we demonstrated that ampicillin resistance was transferable to an E. faecium recipient containing a pbp5 deletion for all but four strains. The transfers occurred at low frequencies (generally ca. 10(-9) transconjugants/recipient CFU), consistent with chromosome-to-chromosome transfer. pbp5 transfer occurred within large genetic regions, and insertion into the recipient genome occurred most commonly into the recipient SmaI restriction fragment that had been created by the previous pbp5 deletion. Restriction mapping of the region upstream of pbp5 revealed a commonality of fragment sizes among the clinical isolates from the United States which differed significantly from those of three strains that were isolated from turkey feces. These data prove conclusively that E. faecium pbp5 is a transferable determinant, even in the absence of a coresiding vancomycin resistance mobile element. They also suggest that the spread of high-level ampicillin resistance among U.S. E. faecium strains is due in part to the transfer of low-affinity pbp5 between clinical isolates.

Tn5386, a novel Tn916-like mobile element in Enterococcus faecium D344R that interacts with Tn916 to yield a large genomic deletion.

We describe Tn5386, a novel ca.-29-kb Tn916-like mobile element discovered to occur in ampicillin-resistant, Tn916-containing Enterococcus faecium D344R. PCR amplification experiments after overnight growth with or without tetracycline revealed "joint" regions of circularized Tn5386 composed of 6-bp sequences linking different transposon termini. In one case (no tetracycline), the termini were consistent with those derived by target site analysis of the integrated element. In the other case, the termini were virtually identical in distance from the integrase binding regions, as seen with Tn916. These data are consistent with a model in which one PCR product results from the action of Tn5386 integrase, whereas the other results from the action of the Tn916 integrase on Tn5386. Spontaneous conversion of D344R to an ampicillin-susceptible phenotype (D344SRF) was associated with a 178-kb deletion extending from the left end of Tn5386 to the left end of Tn916. Examination of the Tn5386 junction after the large deletion event suggests that the deletion resulted from an interaction between the nonintegrase ends of Tn5386 and Tn916. The terminus of Tn5386 identified in this reaction suggested that it may have resulted from the activity of the Tn916 integrase (Int(Tn916)). The "joint" of the circular element resulting from this excision was amplifiable from D344R, the sequence of which revealed a heteroduplex consistent with Int(Tn916)-mediated excision. In contrast, Tn5386 joints amplified from ampicillin-susceptible D344SRF revealed ends consistent with Tn5386 integrase activity, reflecting the absence of Tn916 from this strain. Tn5386 represents a new member of the Tn916 transposon family. Our data suggest that excision of Tn5386 can be catalyzed by the Tn916 integrase and that large genomic deletions may result from the interaction between these heterologous elements.

Impact of specific pbp5 mutations on expression of beta-lactam resistance in Enterococcus faecium.

We tested the impact of individual PBP 5 mutations on expression of ampicillin resistance in Enterococcus faecium using a shuttle plasmid designed to facilitate expression of cloned pbp5 in ampicillin-susceptible E. faecium D344SRF. Substitutions that had been implicated in contributing to the resistance of clinical strains conferred only modest levels of resistance when they were present as single point mutations. The levels of resistance were amplified when some mutations were present in combination. In particular, a methionine-to-alanine change at position 485 (in close proximity to the active site) combined with the insertion of a serine at position 466 (located in a loop that forms the outer edge of the active site) was associated with the highest levels of resistance to all beta-lactams. Affinity for penicillin generally correlated with beta-lactam MICs for the mutants, but these associations were not strictly proportional.