Bleeker-Rovers, C. P. | bioRxiv Science

bioRxiv2021

Multiplex immunohistochemistry differences between Q fever and atherosclerotic abdominal aortic aneurysms indicate immune suppression

BackgroundChronic Q fever is a zoonosis caused by the bacterium Coxiella burnetii which can manifest as infection of an abdominal aortic aneurysm (AAA). Antibiotic therapy often fails, resulting in severe morbidity and high mortality. Whereas previous studies have focused on inflammatory processes in blood, the aim of this study was to investigate local inflammation in aortic tissue. MethodsMultiplex immunohistochemistry was used to investigate local inflammation in Q fever AAAs compared to atherosclerotic AAAs in aorta tissue specimen. Two six-plex panels were used to study both the innate and adaptive immune system. ResultsQ fever AAAs and atherosclerotic AAAs contained similar numbers of CD68+ macrophages and CD3+ T cells. However, in Q fever AAAs the number of CD68+CD206+ M2 macrophages was increased, while expression of GM-CSF was decreased compared to atherosclerotic AAAs. Furthermore, Q fever AAAs showed an increase in both the number of CD8+ cytotoxic T cells and CD3+FoxP3+ regulatory T cells. Lastly, Q fever AAAs did not contain any well-defined granulomas. ConclusionsThese findings demonstrate that despite the presence of pro-inflammatory effector cells, there is an immune suppressive micro environment in Q fever AAA resulting in persistent local infection with C. burnetii.

immunology↗

bioRxiv2020

Novel SARS-CoV-2 Whole-genome sequencing technique using Reverse Complement PCR enables fast and accurate outbreak analysis

BackgroundCurrent transmission rates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are still increasing and many countries are facing second waves of infections. Rapid SARS-CoV-2 whole-genome sequencing (WGS) is often unavailable but could support public health organizations and hospitals in monitoring and determining transmission links. Here we report the use of reverse complement polymerase chain reaction (RC-PCR), a novel technology for WGS of SARS-CoV-2 enabling library preparation in a single PCR saving time, resources and enables high throughput screening. Additionally, we show SARS-CoV-2 diversity and possible transmission within the Radboud university medical center (Radboudumc) during September 2020 using RC-PCR WGS. MethodsA total of 173 samples tested positive for SARS-CoV-2 between March and September 2020 were selected for whole-genome sequencing. Ct values of the samples ranged from 16 to 42. They were collected from 83 healthcare workers and three patients at the Radboudumc, in addition to 64 people living in the area around the hospital and tested by the local health services. For validation purposes, nineteen of the included samples were previously sequenced using Oxford Nanopore Technologies and compared to RC-PCR WGS results. The applicability of RC-PCR WGS in outbreak analysis for public health service and hospitals was tested on six suspected clusters containing samples of healthcare workers and patients with an epidemiological link. FindingsRC-PCR resulted in sequencing data for 146 samples. It showed a genome coverage of up to 98,2% for samples with a maximum Ct value of 32. Comparison to Oxford Nanopore technologies gives a near-perfect agreement on 95% of the samples (18 out of 19). Three out of six clusters with a suspected epidemiological link were fully confirmed, in the others, four healthcare workers were not associated. In the public health service samples, a previously unknown chain of transmission was confirmed. Significance statementSAR-CoV-2 whole-genome sequencing using RC-PCR is a reliable technique and applicable for use in outbreak analysis and surveillance. Its ease of use, high-trough screening capacity and wide applicability makes it a valuable addition or replacement during this ongoing SARS-CoV-2 pandemic. FundingNone Research in contextO_ST_ABSEvidence before this studyC_ST_ABSAt present whole genome sequencing techniques for SARS-CoV-2 have a large turnover time and are not widely available. Only a few laboratories are currently able to perform large scale SARS-CoV-2 sequencing. This restricts the use of sequencing to aid hospital and community infection prevention. Added value of this studyHere we present clinical and technical data on a novel Whole Genome Sequencing technology, implementing reverse-complement PCR. It is able to obtain high genome coverage of SARS-CoV-2 and confirm and exclude epidemiological links in 173 healthcare workers and patients. The RC-PCR technology simplifies the workflow thereby reducing hands on time. It combines targeted PCR and sequence library construction in a single PCR, which normally takes several steps. Additionally, this technology can be used in concordance with the widely available range of Illumina sequencers. Implications of all the available evidenceRC-PCR whole genome sequencing technology enables rapid and targeted surveillance and response to an ongoing outbreak that has great impact on public health and society. Increased use of sequencing technologies in local laboratories can help prevent increase of SARS-CoV-2 spreading by better understanding modes of transmission.

microbiology↗

bioRxiv2019

Coxiella burnetii epitope-specific T-cell responses in chronic Q fever patients

Infection with Coxiella burnetii, the causative agent of Q fever, can result in life-threatening persistent infection. Reactogenicity hinders worldwide implementation of the only licensed human Q fever vaccine. We previously demonstrated long-lived immunoreactivity in individuals with past symptomatic and asymptomatic Coxiella infection (convalescents) to promiscuous HLA-class II C. burnetii epitopes, providing the basis for a novel T-cell-targeted subunit vaccine. Here we investigated in a cohort of 22 individuals with persistent infection (chronic Q fever) whether they recognize the same set of epitopes, or distinct epitopes that could be candidates for a therapeutic vaccine or aid in the diagnosis of persistent infection.\n\nIndividuals with chronic Q fever showed strong class II epitope-specific cultured ELISpot responses largely overlapping with the peptide repertoire identified previously for convalescents. Five additional peptides were recognized more frequently by chronic subjects, but there was no combination of epitopes uniquely recognized by or non-reactive in chronic Q fever subjects. Consistent with more recent/prolonged exposure, we found, however, stronger direct ex vivo responses to whole-cell C. burnetii and individual peptides in direct ELISpot than in convalescents.\n\nIn conclusion, we have validated and expanded a previously published set candidate epitopes for a novel T-cell targeted subunit Q fever vaccine in the context of chronic Q fever patients and demonstrated that they successfully mounted a T-cell response comparable to that of convalescents. Finally, we demonstrate that individuals treated for chronic Q fever mount a broader ex vivo response to class II epitopes than convalescents, which could be explored for diagnostic purposes.

immunology↗