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

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

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

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

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

The KQ Element, a Complex Genetic Region Conferring Transferable Resistance to Carbapenems, Aminoglycosides, and Fluoroquinolones in Klebsiella pneumoniae▿

The blaKPC-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