BurkPx: a multiplex serodiagnostic bead assay to detect Burkholderia pseudomallei exposures in non-human primates

Melioidosis is a fatal zoonotic disease caused by the soil-dwelling Gram-negative bacteria Burkholderia pseudomallei. It is most well-known for its burden on humans, with an estimated 165,000 human cases per year worldwide, but this illness is equally important for animals, including non-human primates (NHPs). Although there is no published disease burden for NHPs we can look at the handful of reported melioidosis cases dating back to the 1920’s in multiple countries to make the conclusion that burden is not great, but possibly sporadic. Currently the gold standard for detecting B. pseudomallei in humans and animals is to culture from clinical specimens; however, the sensitivity of this technique is subpar. An alternative test, the indirect hemagglutination assay (IHA) is the standard serological assay for melioidosis, but also has low metrics. The search for a rapid, highly sensitive and specific assay is ongoing. Both direct detection methods like PCR and indirect detection methods like enzyme linked absorbent assays have been developed and evaluated. We have developed a multiplexed serological assay, called BurkPx, which uses purified B. pseudomallei proteins and carbohydrates bound to 21 fluorescently distinct MagPix magnetic beads to standardize a serodiagnostic assay for NHP. We screened B. pseudomallei-challenged (n=115) and non-challenged (n=126) rhesus macaques to explore time-dependent antibody responses and to train two multivariate models, LASSO and Ridge Regression, in differentiating exposure from non-exposure. When cross-validating the model using independent data, 59.6% sensitivity and 100% specificity was observed. However, removal of week 1 samples increased the sensitivity to 91.3%. To our knowledge, this is the first diagnostic tool, a serological multiplex assay, to be formally evaluated for NHPs and results in promising sensitivity and specificity metrics

Genomic characterization of Francisella tularensis and other diverse Francisella species from complex samples

Francisella tularensis, the bacterium that causes the zoonosis tularemia, and its genetic near neighbor species, can be difficult or impossible to cultivate from complex samples. Thus, there is a lack of genomic information for these species that has, among other things, limited the development of robust detection assays for F. tularensis that are both specific and sensitive. The objective of this study was to develop and validate approaches to capture, enrich, sequence, and analyze Francisella DNA present in DNA extracts generated from complex samples. RNA capture probes were designed based upon the known pan genome of F. tularensis and other diverse species in the family Francisellaceae. Probes that targeted genomic regions also present in non-Francisellaceae species were excluded, and probes specific to particular Francisella species or phylogenetic clades were identified. The capture-enrichment system was then applied to diverse, complex DNA extracts containing low-level Francisella DNA, including human clinical tularemia samples, environmental samples (i.e., animal tissue and air filters), and whole ticks/tick cell lines, which was followed by sequencing of the enriched samples. Analysis of the resulting data facilitated rigorous and unambiguous confirmation of the detection of F. tularensis or other Francisella species in complex samples, identification of mixtures of different Francisella species in the same sample, analysis of gene content (e.g., known virulence and antimicrobial resistance loci), and high-resolution whole genome-based genotyping. The benefits of this capture-enrichment system include: even very low target DNA can be amplified; it is culture-independent, reducing exposure for research and/or clinical personnel and allowing genomic information to be obtained from samples that do not yield isolates; and the resulting comprehensive data not only provide robust means to confirm the presence of a target species in a sample, but also can provide data useful for source attribution, which is important from a genomic epidemiology perspective

Diverse lineages of pathogenic Leptospira species are widespread in the environment in Puerto Rico, USA.

BackgroundLeptospirosis, caused by Leptospira bacteria, is a common zoonosis worldwide, especially in the tropics. Reservoir species and risk factors have been identified but surveys for environmental sources are rare. Furthermore, understanding of environmental Leptospira containing virulence associated genes and possibly capable of causing disease is incomplete, which may convolute leptospirosis diagnosis, prevention, and epidemiology.Methodology/principal findingsWe collected environmental samples from 22 sites in Puerto Rico during three sampling periods over 14-months (Dec 2018-Feb 2020); 10 water and 10 soil samples were collected at each site. Samples were screened for DNA from potentially pathogenic Leptospira using the lipL32 PCR assay and positive samples were sequenced to assess genetic diversity. One urban site in San Juan was sampled three times over 14 months to assess persistence in soil; live leptospires were obtained during the last sampling period. Isolates were whole genome sequenced and LipL32 expression was assessed in vitro. We detected pathogenic Leptospira DNA at 15/22 sites; both soil and water were positive at 5/15 sites. We recovered lipL32 sequences from 83/86 positive samples (15/15 positive sites) and secY sequences from 32/86 (10/15 sites); multiple genotypes were identified at 12 sites. These sequences revealed significant diversity across samples, including four novel lipL32 phylogenetic clades within the pathogenic P1 group. Most samples from the serially sampled site were lipL32 positive at each time point. We sequenced the genomes of six saprophytic and two pathogenic Leptospira isolates; the latter represent a novel pathogenic Leptospira species likely belonging to a new serogroup.Conclusions/significanceDiverse and novel pathogenic Leptospira are widespread in the environment in Puerto Rico. The disease potential of these lineages is unknown but several were consistently detected for >1 year in soil, which could contaminate water. This work increases understanding of environmental Leptospira diversity and should improve leptospirosis surveillance and diagnostics

Factor Xa cleaves SARS-CoV-2 spike protein to block viral entry and infection

The serine protease factor Xa (FXa) is upregulated in COVID-19 patients and functions in the coagulation pathway. Here, Dong et al characterise the basis of its antiviral activity in the context of SARS-CoV-2 pandemic variants

COVID-19 vaccination elicits an evolving, cross-reactive antibody response to epitopes conserved with endemic coronavirus spike proteins.

The COVID-19 pandemic has triggered the first widespread vaccination campaign against a coronavirus. Many vaccinated subjects are previously naive to SARS-CoV-2; however, almost all have previously encountered other coronaviruses (CoVs), and the role of this immunity in shaping the vaccine response remains uncharacterized. Here, we use longitudinal samples and highly multiplexed serology to identify mRNA-1273 vaccine-induced antibody responses against a range of CoV Spike epitopes, in both phylogenetically conserved and non-conserved regions. Whereas reactivity to SARS-CoV-2 epitopes shows a delayed but progressive increase following vaccination, we observe distinct kinetics for the endemic CoV homologs at conserved sites in Spike S2: these become detectable sooner and decay at later time points. Using homolog-specific antibody depletion and alanine-substitution experiments, we show that these distinct trajectories reflect an evolving cross-reactive response that can distinguish rare, polymorphic residues within these epitopes. Our results reveal mechanisms for the formation of antibodies with broad reactivity against CoVs