Validation of a Methodology for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2 in Saliva by Real-Time Reverse Transcriptase-PCR.

In January 2021, the Chilean city of Concepción experienced a second wave of coronavirus 2019 (COVID-19) while in early April 2021, the entire country faced the same situation. This outbreak generated the need to modify and validate a method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in saliva, thereby expanding the capacity and versatility of testing for COVID-19. This study was conducted in February 2021 in the Chilean city of Concepción during which time, the town was under total quarantine. The study participants were mostly symptomatic (87.4%), not hospitalized, and attended care centers because of their health status rather than being asked by the researchers. People coming to the health center in Concepción to be tested for COVID-19 (via reverse transcriptase polymerase chain reaction [RT-PCR]) from a specimen of nasopharyngeal swab (NPS) were then invited to participate in this study. A total of 131 participants agreed to sign an informed consent and to provide saliva and NPS specimens to validate a method in terms of sensitivity, specificity, and statistical analysis of the cycle threshold (Ct) values from the RT-PCR. Calculations pertaining to the 127 participants who were ultimately included in the analysis showed sensitivity and specificity at 94.34% (95% CI: 84.34-98.82%) and 98.65% (95% CI: 92.70-99.97%), respectively. The saliva specimen showed a performance comparable to NPS as demonstrated by the diagnostic parameters. This RT-PCR method from the saliva specimen is a highly sensitive and specific alternative compared to the reference methodology, which uses the NPS specimen. This modified and validated method is intended for use in the in vitro diagnosis of SARS-CoV-2, which provides health authorities in Chile and local laboratories with a real testing alternative to RT-PCR from NPS.

Development and analytical validation of real-time PCR for the detection of Streptococcus agalactiae in pregnant women.

BACKGROUND: Group B Streptococcus (GBS) is the leading cause of invasive neonatal infection. In this study, we aimed to evaluate the analytical validation of qualitative real-time polymerase chain reaction (qPCR) as a means to detect GBS. METHODS: Genomic DNA (gDNA) was purified from 12 ATCC bacterial strains, two belonging to GBS and the remainder acting as negative controls. Additionally, gDNA was isolated from 21 strains of GBS from various serotypes (Ia, Ib and II-VIII). All gDNA was used to evaluate the analytical validation of the qPCR method employing a specific Taqman probe. Inclusivity, exclusivity, anticipated reportable range, the limit of detection and robustness were evaluated. The methods used are described in international guidelines and other existing reports. The performance of this qPCR method for detecting GBS was compared to other microbiological methods used with vaginal-rectal samples from pregnant women. RESULTS: Our qPCR method for detecting GBS was analytically validated. It has a limit of detection of 0.7 GE/µL and 100% analytical specificity. It detects all strains of GBS with the same level of performance as microbiological methods. CONCLUSION: Data suggest that this qPCR method performs adequately as a means to detect GBS in vaginal-rectal swabs from pregnant women.

Mucosal Vaccination with Lactococcus lactis-Secreting Surface Immunological Protein Induces Humoral and Cellular Immune Protection against Group B Streptococcus in a Murine Model.

Group B Streptococcus (GBS) is the primary etiological agent of sepsis and meningitis in newborns and is associated with premature birth and stillbirth. The development of a licensed vaccine is one of the pending challenges for the World Health Organization. Previously, we showed that oral immunization with surface immune protein (SIP) decreases vaginal colonization of GBS and generates functional opsonizing antibodies, which was determined by opsonophagocytic assays (OPA) in vitro. We also showed that the protein has an adjuvant vaccine profile. Therefore, an oral vaccine based on SIP may be an attractive alternative to employ in the development of new vaccines against GBS. Lactococcus lactis is a highlighted oral vaccine probiotic inducer of the mucosal immune response. This bacterium could serve as an antigen-delivering vehicle for the development of an edible vaccine and has been used in clinical trials. In this study, we showed that an oral vaccine with a recombinant L. lactis strain secreting SIP from GBS (rL. lactis-SIP) can induce protective humoral and cellular immunity in an experimental model of GBS vaginal colonization in C57BL/6 mice. Mice immunized with rL. lactis-SIP were protected against clinical symptoms and bacterial colonization after GBS vaginal colonization. Our rL. lactis-SIP vaccine also induces an increase of immunoglobulin G (IgG) and immunoglobulin A (IgA) specifically against SIP. The adoptive transfer of serum from vaccinated mice to naïve mice generated protection against GBS vaginal colonization. Moreover, the rL. lactis-SIP strain induces the activation of SIP-specific T cells, which could decrease GBS vaginal colonization and generate protective antibodies when transferred to other mice. Our experimental observations strongly support the notion that rL. lactis-SIP induces protective humoral and cellular immunity and could be considered as a novel alternative in the development of vaccines for GBS.

Surface Immunogenic Protein of Streptococcus Group B is an Agonist of Toll-Like Receptors 2 and 4 and a Potential Immune Adjuvant.

Vaccine-induced protection against pathogens, especially subunit-based vaccines, are related to antigen properties but mainly in their ability to stimulate the immune system by the use of an adjuvant. Modern vaccines are formulated with a high level of antigen purity, where an efficient adjuvant is necessary. In this context, the use of protein Toll-Like Receptor (TLR) agonists as vaccine adjuvants has been highlighted because of their optimal immunogenicity and minimal toxicity. The Surface Immunogenic Protein (SIP) from Group B Streptococcus (GBS) has gained importance as a new potential protein-based vaccine. Recently, we reported that recombinant SIP (rSIP) expressed by E. coli and purified by High Performance Liquid Chromatography (HPLC) alone induces a protective humoral immune response. In this study, we present the immunomodulatory properties of rSIP as a protein-based adjuvant, as an agonist of TLR. To this end, we showed that C57BL/6 bone marrow-derived dendritic cells pulsed by rSIP resulted in enhanced CD40, CD80, CD86, and Major Histocompatibility Complex (MHC) class II as well as increased secretion proinflammatory cytokines Interleukin (IL)-6, Interferon (IFN)-γ, Tumor Necrosis Factor (TNF)-α, and IL-10. Next, we investigated the in vivo effect of rSIP in the absence or presence of ovalbumin (OVA) on antigen-specific antibody secretion in C57BL/6 mice. Immunization with rSIP plus OVA showed that anti-OVA IgG2a and IgG1a increased significantly compared with OVA alone in C57BL/6 mice. Also, the immunization of rSIP plus OVA generates increased serum cytokines levels characterized by IL-12p70, IL-10, IL-4, and IFN-γ. Interestingly, we observed that rSIP stimulate Toll Like Receptor (TLR)2 and TLR4, individually expressed by Human embryonic kidney (HEK) 293-derived TLR reporter cells. These findings suggest that rSIP is a new potential protein TLR agonist adjuvant and may be employed in the development of new vaccines.

Cellular immune response induced by surface immunogenic protein with AbISCO-100 adjuvant vaccination decreases group B Streptococcus vaginal colonization.

Group B Streptococcus (GBS) represents one of the most common causes of bacterial infection in neonates; it is also associated with premature childbirth and stillbirth. A vaccine against GBS is needed, but no approved vaccines are yet available. The Surface Immunogenic Protein (SIP) of GBS is conserved in all serotypes and had been reported to be a good vaccine prototype in a mouse model of GBS infection. Also, we have previously shown that both subcutaneous and oral immunization with rSIP can induce an efficient immune response that decreases GBS vaginal colonization in mice. In this study, we show that a vaccine based on a mixture of rSIP and AbISCO-100 adjuvant reduces GBS vaginal colonization in mice and induces antibodies with opsonophagocytic activities. Moreover, the passive transfer of sera and total T-cells from mice immunized with rSIP mixed with AbISCO-100 to unvaccinated mice decreases vaginal GBS colonization in an infected mouse. This is the first report of cellular immunity associated with rSIP-based vaccine testing in a mouse model of GBS infection.

Virtual Coronary Intervention: A Treatment Planning Tool Based Upon the Angiogram.

OBJECTIVES: This study sought to assess the ability of a novel virtual coronary intervention (VCI) tool based on invasive angiography to predict the patient's physiological response to stenting. BACKGROUND: Fractional flow reserve (FFR)-guided percutaneous coronary intervention (PCI) is associated with improved clinical and economic outcomes compared with angiographic guidance alone. Virtual (v)FFR can be calculated based upon a 3-dimensional (3D) reconstruction of the coronary anatomy from the angiogram, using computational fluid dynamics (CFD) modeling. This technology can be used to perform virtual stenting, with a predicted post-PCI FFR, and the prospect of optimized treatment planning. METHODS: Patients undergoing elective PCI had pressure-wire-based FFR measurements pre- and post-PCI. A 3D reconstruction of the diseased artery was generated from the angiogram and imported into the VIRTUheart workflow, without the need for any invasive physiological measurements. VCI was performed using a radius correction tool replicating the dimensions of the stent deployed during PCI. Virtual FFR (vFFR) was calculated pre- and post-VCI, using CFD analysis. vFFR pre- and post-VCI were compared with measured (m)FFR pre- and post-PCI, respectively. RESULTS: Fifty-four patients and 59 vessels underwent PCI. The mFFR and vFFR pre-PCI were 0.66 ± 0.14 and 0.68 ± 0.13, respectively. Pre-PCI vFFR deviated from mFFR by ±0.05 (mean Δ = -0.02; SD = 0.07). The mean mFFR and vFFR post-PCI/VCI were 0.90 ± 0.05 and 0.92 ± 0.05, respectively. Post-VCI vFFR deviated from post-PCI mFFR by ±0.02 (mean Δ = -0.01; SD = 0.03). Mean CFD processing time was 95 s per case. CONCLUSIONS: The authors have developed a novel VCI tool, based upon the angiogram, that predicts the physiological response to stenting with a high degree of accuracy.

The Optimisation of the Expression of Recombinant Surface Immunogenic Protein of Group B Streptococcus in Escherichia coli by Response Surface Methodology Improves Humoral Immunity.

Group B Streptococcus (GBS) is the leading cause of neonatal meningitis and a common pathogen in livestock and aquaculture industries around the world. Conjugate polysaccharide and protein-based vaccines are under development. The surface immunogenic protein (SIP) is a conserved protein in all GBS serotypes and has been shown to be a good target for vaccine development. The expression of recombinant proteins in Escherichia coli cells has been shown to be useful in the development of vaccines, and the protein purification is a factor affecting their immunogenicity. The response surface methodology (RSM) and Box-Behnken design can optimise the performance in the expression of recombinant proteins. However, the biological effect in mice immunised with an immunogenic protein that is optimised by RSM and purified by low-affinity chromatography is unknown. In this study, we used RSM for the optimisation of the expression of the rSIP, and we evaluated the SIP-specific humoral response and the property to decrease the GBS colonisation in the vaginal tract in female mice. It was observed by NI-NTA chromatography that the RSM increases the yield in the expression of rSIP, generating a better purification process. This improvement in rSIP purification suggests a better induction of IgG anti-SIP immune response and a positive effect in the decreased GBS intravaginal colonisation. The RSM applied to optimise the expression of recombinant proteins with immunogenic capacity is an interesting alternative in the evaluation of vaccines in preclinical phase, which could improve their immune response.

Immunization with a Mixture of Nucleoprotein from Human Metapneumovirus and AbISCO-100 Adjuvant Reduces Viral Infection in Mice Model.

The human metapneumovirus (hMPV) is the second leading cause globally of acute infection of the respiratory tract in children, infecting the upper and lower airways. The hMPV may induce an inappropriate Th2-type immune response, which causes severe pulmonary inflammation, leading to the obstruction of airways. Despite its severe epidemiological relevance, no vaccines are currently available for the prevention of hMPV-induced illness. In this investigation, we demonstrated that immunization of mice with the recombinant hMPV nucleoprotein (hMPV-N) mixed with the AbISCO-100 adjuvant reduced viral replication in lungs following challenge with the virus. We found that immunized mice had reduced weight loss, decreased granulocytes in the lung, an increased level of specific nucleoprotein antibodies of IgG1 and IgG2a-isotypes, and a local profile of Th1/Th17-type cytokines. Our results suggest that immunization with the hMPV-N and the AbISCO-100 adjuvant induces a reduction of viral infection and could be considered for the development of an hMPV vaccine.

Oral administration of recombinant Neisseria meningitidis PorA genetically fused to H. pylori HpaA antigen increases antibody levels in mouse serum, suggesting that PorA behaves as a putative adjuvant.

The Neisseria meningitidis outer membrane protein PorA from a Chilean strain was purified as a recombinant protein. PorA mixed with AbISCO induced bactericidal antibodies against N. meningitidis in mice. When PorA was fused to the Helicobacter pylori HpaA antigen gene, the specific response against H. pylori protein increased. Splenocytes from PorA-immunized mice were stimulated with PorA, and an increase in the secretion of IL-4 was observed compared with that of IFN-γ. Moreover, in an immunoglobulin sub-typing analysis, a substantially higher IgG1 level was found compared with IgG2a levels, suggesting a Th2-type immune response. This study revealed a peculiar behavior of the purified recombinant PorA protein per se in the absence of AbISCO as an adjuvant. Therefore, the resistance of PorA to proteolytic enzymes, such as those in the gastrointestinal tract, was analyzed, because this is an important feature for an oral protein adjuvant. Finally, we found that PorA fused to the H. pylori HpaA antigen, when expressed in Lactococcus lactis and administered orally, could enhance the antibody response against the HpaA antigen approximately 3 fold. These observations strongly suggest that PorA behaves as an effective oral adjuvant.