Engineering of DNA Structures Attached to Magnetic Particles for Effective Trans- and Cis-Cleavage in Cas12-Based Biosensors.

Sequence-specific endonuclease Cas12-based biosensors have rapidly evolved as a strong tool to detect nucleic acids. Magnetic particles (MPs) with attached DNA structures could be used as a universal platform to manipulate the DNA-cleavage activity of Cas12. Here, we propose nanostructures of trans- and cis-DNA targets immobilized on the MPs. The main advantage of the nanostructures is a rigid double-stranded DNA adaptor that distances the cleavage site from the MP surface to ensure maximum Cas12 activity. Adaptors with different lengths were compared by detecting the cleavage by fluorescence and gel electrophoresis of the released DNA fragments. The length-dependent effects for cleavage on the MPs' surface were found both for cis- and trans-targets. For trans-DNA targets with a cleavable 15-dT tail, the results showed that the optimal range of the adaptor length was 120-300 bp. For cis-targets, we varied the length and location of the adaptor (at the PAM or spacer ends) to estimate the effect of the MP's surface on the PAM-recognition process or R-loop formation. The sequential arrangement of an adaptor, PAM, and a spacer was preferred and required the minimum adaptor length of 3 bp. Thus, with cis-cleavage, the cleavage site can be located closer to the surface of the MPs than with trans-cleavage. The findings provide solutions for efficient Cas12-based biosensors using surface-attached DNA structures.

Rapid Detection of Lipopolysaccharide and Whole Cells of Francisella tularensis Based on Agglutination of Antibody-Coated Gold Nanoparticles and Colorimetric Registration.

The paper presents development and characterization of a new bioanalytical test system for rapid detection of lipopolysaccharide (LPS) and whole cells of Francisella tularensis, a causative agent of tularemia, in water samples. Gold nanoparticles (AuNPs) coated by the obtained anti-LPS monoclonal antibodies were used for the assay. Their contact with antigen in tested samples leads to aggregation with a shift of absorption spectra from red to blue. Photometric measurements at 530 nm indicated the analyte presence. Three preparations of AuNPs with different diameters were compared, and the AuNPs having average diameter of 34 nm were found to be optimal. The assay is implemented in 20 min and is characterized by detection limits equal to 40 ng/mL for LPS and 3 × 104 CFU/mL for whole cells of F. tularensis. Thus, the proposed simple one-step assay integrates sensitivity comparable with other immunoassay of microorganisms and rapidity. Selectivity of the assay for different strains of F. tularensis was tested and the possibility to choose its variants with the use of different antibodies to distinguish virulent and non-virulent strains or to detect both kinds of F. tularensis was found. The test system has been successfully implemented to reveal the analyte in natural and tap water samples without the loss of sensitivity.

Lateral Flow Immunoassay of SARS-CoV-2 Antigen with SERS-Based Registration: Development and Comparison with Traditional Immunoassays.

The current COVID-19 pandemic has increased the demand for pathogen detection methods that combine low detection limits with rapid results. Despite the significant progress in methods and devices for nucleic acid amplification, immunochemical methods are still preferred for mass testing without specialized laboratories and highly qualified personnel. The most widely used immunoassays are microplate enzyme-linked immunosorbent assay (ELISA) with photometric detection and lateral flow immunoassay (LFIA) with visual results assessment. However, the disadvantage of ELISA is its considerable duration, and that of LFIA is its low sensitivity. In this study, the modified LFIA of a specific antigen of the causative agent of COVID-19, spike receptor-binding domain, was developed and characterized. This modified LFIA includes the use of gold nanoparticles with immobilized antibodies and 4-mercaptobenzoic acid as surface-enhanced Raman scattering (SERS) nanotag and registration of the nanotag binding by SERS spectrometry. To enhance the sensitivity of LFIA-SERS analysis, we determined the optimal compositions of SERS nanotags and membranes used in LFIA. For benchmark comparison, ELISA and conventional colorimetric LFIA were used with the same immune reagents. The proposed method combines a low detection limit of 0.1 ng/mL (at 0.4 ng/mL for ELISA and 1 ng/mL for qualitative LFIA) with a short assay time equal to 20 min (at 3.5 h for ELISA and 15 min for LFIA). The results obtained demonstrate the promise of using the SERS effects in membrane immuno-analytical systems.

Comparative Study of In Situ Techniques to Enlarge Gold Nanoparticles for Highly Sensitive Lateral Flow Immunoassay of SARS-CoV-2.

Three techniques were compared for lowering the limit of detection (LOD) of the lateral flow immunoassay (LFIA) of the receptor-binding domain of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) based on the post-assay in situ enlargement of Au nanoparticles (Au NPs) on a test strip. Silver enhancement (growth of a silver layer over Au NPs-Au@Ag NPs) and gold enhancement (growth of a gold layer over Au NPs) techniques and the novel technique of galvanic replacement of Ag by Au in Au@Ag NPs causing the formation of Au@Ag-Au NPs were performed. All the enhancements were performed on-site after completion of the conventional LFIA and maintained equipment-free assay. The assays demonstrated lowering of LODs in the following rows: 488 pg/mL (conventional LFIA with Au NPs), 61 pg/mL (silver enhancement), 8 pg/mL (galvanic replacement), and 1 pg/mL (gold enhancement). Using gold enhancement as the optimal technique, the maximal dilution of inactivated SARS-CoV-2-containing samples increased 500 times. The developed LFIA provided highly sensitive and rapid (8 min) point-of-need testing.

Immunological markers that correlate with protection immunity against tularemia infection.

An efficient immune response to tularemia is dependent on a strong cell-mediated component. We tried to identify markers of cellular immune responses that indicate a vaccine efficacy against tularemia. BALB/c mice were immunized with mutant F. tularensis 15∆23A and/or F. tularensis 15 NIIEG strains and then were challenged i.n. with F. tularensis Schu. We compared the influence of F. tularensis antigens (tularinum) in vitro on production of IL-1, IL-5, IL-6, IL-17, IFN-γ, and TNF-α by splenocytes obtained from intact mice and mice immunized with mutant F. tularensis 15∆23A and/or F. tularensis 15 NIIEG strains. We also compared expression of CD28, CD154, TLR-2, and CD69 markers on CD4 and CD8 T-cells after activation with tularinum in vitro. We found that tularinum-induced CD4(+) T-cells increased TNF-α and IFN-γ synthesis and expression of CD69 only in group mice with high degree of post immunization protection against F. tularensis Schu challenge. Estimation of CD69 expression on CD3(+)CD4(+) cells and IFN-γ, TNF-α synthesis by CD4(+) T-lymphocytes could be useful for determination protect ability of antitularemia immunity.

Rational design of antigens to improve the serodiagnosis of tick-borne borreliosis in central regions of Russia.

Tick-borne borreliosis (Lyme disease-LD) is caused by pathogenic Borrelia spirochetes that is transmitted through bite of Ixodes ticks to humans and animals. In the Russian Federation, borreliosis registered with an index of 6-7 per 100,000 people annually. In reality, LD morbidity in Russia is much higher because Russian strains develop less erythematous rashes compared to North American strains, thus missed by physicians in most of the early cases, and current serology tests have insufficient sensitivity as well. The aim of this work was to improve the sensitivity and specificity of serology tests for LD in Russia using rationale-designed Borrelia antigens. It was anticipated that sensitivity of LD sero-diagnosis will be higher if antigen for test-systems are derived from a strain that is circulated in a geographical region of test application. A large portion of the Russian population lives in the Central region. Thus, effort has been made to create a serological test using antigens from Moscow region, Tula and Ul'janovsk areas. In this study we included wild strains (ultrasonic-treated spirochetes B. garinii H19, B. afzelii P1, B. afzelii P1H13, B. burgdorferi s.s. 39/40, B. burgdorferi s.s. B31), recombinant (expressed in E.coli DbpA, Bgp, Bbk B. garinii, and B. afzelii) antigens and some of their combinations were produced and tested against LD patients and donors serum collected in hospitals of Central regions of Russia by ELISA and Western blotting. Considering sensitivity and specificity, DbpA B. afzelii and DbpA B. garinii recombinant antigens were selected among all probed antigens for regional serology test. As long as DbpA B. afzelii and DbpA B. garinii antigens interacted with LD patient's serum in a complementary mode, it is possible to combine epitopes DbpA B. afzelii and B. garinii in a single antigen for improving sensitivity. We created recombinant fusion protein DbpA B. afzelii/B using dbpA genes from Russian isolates of B. afzelii and B. garinii in E. coli. Fusion DbpA A + G protein was then used for formulation of fast immunochromatographic serodiagnosis test (LF) in a "deep-stick" format. The trials of LF-test were conducted separately at Institute of Rheumatology Russian Academy of Medical Science (using 325 sera) and at the Borreliosis Reference Center of Ministry of Health RF (using 120 reference sera). The average sensitivity and specificity of LF-test was 80.5 and 100 %, respectively.

Mycobacterium tuberculosis attenuated by multiple deletions of rpf genes effectively protects mice against TB infection.

In this study, we investigated the residual virulence of mutants of Mycobacterium tuberculosis that are defective in 4 of the 5 rpf-like genes, their capacity to persist in the murine host and the utility present in these mutants to serve as novel vaccine candidates. Our data indicate that the two quadruple rpf deletion mutants, ΔACBD and ΔACDE, both display significant attenuation in the mouse lungs after aerosol infection, with no observable increase in bacillary loads upon aminoguanidine-induced immune suppression. However, after subcutaneous injection these strains were able to persist at the low level, similar to that of BCG, in the mouse lungs and lymphoid organs. Furthermore, both rpf quadruple mutants were able to enhance the numbers of IFN-γ-producing T-cells in spleens to a level comparable to that of BCG, and conferred protection upon subsequent challenge with virulent M. tuberculosis in terms of mycobacterial multiplication in organs and survival time. The reduction in organ bacillary loads after vaccination with ΔACDE was comparable to that of BCG, while ΔACBD displayed a small but statistically significant enhancement in protection compared to BCG. Collectively, these data suggest that rpf deletion mutants show potential for further development as novel vaccine candidates for tuberculosis.

Molecular identification of Salp15, a key salivary gland protein in the transmission of lyme disease spirochetes, from Ixodes persulcatus and Ixodes pacificus (Acari: Ixodidae).

Salp15 is a multifunctional protein, vital to the tick in its need to obtain vertebrate host blood without stimulating a host inflammatory and immune response. The Salpl5 protein from both Ixodes scapularis Say and Ixodes ricinus (L.), the principal vectors of the Lyme disease spirochete in eastern North America and Europe, respectively, have been well characterized and found to bind the murine CD4 receptor, DC-SIGN, and the OspC protein of Borrelia burgdorferi. In the current study, we characterized the full salp15 gene in Ixodes pacificus Cooley & Kohls and Ixodes persulcatus Schulze, the principal vectors of Lyme disease spirochetes in western North America and Asia, respectively. In comparing the Salp15 protein of all four principal vector ticks of public health importance for the transmission of Lyme disease spirochetes, we find the 53 C-terminal amino acids to have a high degree of similarity. There are at least three clades in the tree of Salp15 and its homologues, probably representing a multigene family.

The role of resuscitation promoting factors in pathogenesis and reactivation of Mycobacterium tuberculosis during intra-peritoneal infection in mice.

BACKGROUND: Mycobacterium tuberculosis can enter into a dormant state which has resulted in one third of the world's population being infected with latent tuberculosis making the study of latency and reactivation of utmost importance. M. tuberculosis encodes five resuscitation promoting factors (Rpfs) that bear strong similarity to a lysozyme-like enzyme previously implicated in reactivation of dormant bacteria in vitro. We have developed an intraperitoneal infection model in mice, with immune modulation, that models chronic infection with similar properties in mouse lungs as those observed in the murine aerosol infection model. We have assessed the behavior of mutants that lack two or three rpf genes in different combinations in our intraperitoneal model. METHODS: C57Bl/6 mice were intraperitonealy infected with H37Rv wild type M. tuberculosis or mutant strains that lacked two or three rpf genes in different combinations. After 90 days of infection aminoguanidine (AG) or anti-TNFalpha antibodies were administrated. Organ bacillary loads were determined at various intervals post infection by plating serial dilutions of organ homogenates and enumerating bacteria. RESULTS: We found that the rpf triple and double mutants tested were attenuated in their ability to disseminate to mouse lungs after intraperitoneal administration and were defective in their ability to re-grow after immunosuppression induced by administration of aminoguanidine and anti-TNFalpha antibodies. CONCLUSION: Rpf proteins may have a significant physiological role for development of chronic TB infection and its reactivation in vivo.