METHODS: In order to investigate the efficacy of pre-clinical vaccine candidates in P. knowlesi-infected human cases, this study describes an in vitro invasion inhibition assay, using a P. knowlesi strain adapted to in vitro growth in human erythrocytes, PkA1-H.1. Recombinant proteins of P. knowlesi Duffy binding protein alpha (PkDBPα) and apical membrane antigen 1 (PkAMA1) were produced in Escherichia coli system and rabbit antibodies were generated from immune animals.
RESULTS: PkDBPα and PkAMA1 recombinant proteins were expressed as insoluble and produced as a functional refolded form for this study. Antibodies against PkDBPα and PkAMA1 specifically recognized recombinant proteins and native parasite proteins in schizont-stage parasites on the merozoite organelles. Single and combination of anti-PkDBPα and anti-PkAMA1 antibodies elicited strong growth inhibitory effects on the parasite in concentration-dependent manner. Meanwhile, IgG prevalence of PkDBPα and PkAMA1 were observed in 13.0 and 46.7% in human clinical patients, respectively.
CONCLUSION: These data provide support for the validation of in vitro growth inhibition assay using antibodies of DBPα and AMA1 in human-adapted P. knowlesi parasite PkA1-H.1 strain.
STUDY DESIGN: Retrospective cohort study using data submitted prospectively to the Malaysian National Neonatal Registry (MNNR).
SETTING: 44 Malaysian NICUs.
PARTICIPANTS: All neonates born in 2015- 2020.
RESULTS: EOS was reported in 991 neonates. The annual incidence of EOS increased from 0.46 to 0.49/1000 livebirths over the six years. The most common pathogen was Streptococcus agalactiae or Group B haemolytic streptococcus (GBS) (n=388, 39.2%), followed by Escherichia coli (E. coli) (n=80, 8.1%), Klebsiella spp (n=73, 7.4%), coagulase negative staphylococcus (CONS) (n=73, 7.4%), Pseudomonas spp (n=44, 4.4%) and methicillin-sensitive Staphylococcus aureus (n=34, 3.4%). The incidence of EOS due to GBS increased from 0.17 to 0.22/1000 livebirths. Morbidities and mortality were higher in those with EOS than without EOS. Multiple logistic regression analysis showed that Indian ethnic group, chorioamnionitis, gestation≥37weeks, female, spontaneous vaginal delivery, instrumental delivery, and surfactant therapy were significantly associated with increased risk of EOS due to GBS. Four factors were significantly associated with increased risk of non-GBS EOS (outborns, birthweight lt;1000 g, vaginal delivery, and surfactant therapy). Early continuous positive airway pressure was associated with significantly lower risk of EOS.
CONCLUSION: The incidence of EOS showed an increasing trend in Malaysian NICUs. GBS was the most common causative pathogen. Several modifiable risk factors associated with EOS have been identified.
METHODS: In this study, new binders derived from shark VNAR domains library were investigated. Following immunization of a wobbegong shark (Orectolobus ornatus) with three recombinant malaria biomarker proteins (PfHRP2, PfpLDH and Pvaldolase), a single domain antibody (sdAb) library was constructed from splenocytes. Target-specific VNAR phage were isolated by panning. One specific clone was selected for expression in Escherichia coli expression system, and study of binding reactivity undertaken.
RESULTS: The primary VNAR domain library possessed a titre of 1.16 × 106 pfu/mL. DNA sequence analysis showed 82.5% of isolated fragments appearing to contain an in-frame sequence. After multiple rounds of biopanning, a highly dominant clone specific to PfHRP2 was identified and selected for protein production in an E. coli expression system. Biological characterization showed the recombinant protein expressed in periplasmic has better detection sensitivity than that of cytoplasmic proteins. Assays of binding activity indicated that its reactivity was inferior to the positive control mAb C1-13.
CONCLUSIONS: Target-specific bacteriophage VNARs were successfully isolated after a series of immunization, demonstrating that phage display technology is a useful tool for selection of antigen binders. Generation of new binding reagents such as VNAR antibodies that specifically recognize the malaria biomarkers represents an appealing approach to improve the performance of RDTs.