IgG and IgM antibody formation to spike and nucleocapsid proteins in COVID-19 characterized by multiplex immunoblot assays.

BACKGROUND: Rapid and simple serological assays for characterizing antibody responses are important in the current COVID-19 pandemic caused by SARS-CoV-2. Multiplex immunoblot (IB) assays termed COVID-19 IB assays were developed for detecting IgG and IgM antibodies to SARS-CoV-2 virus proteins in COVID-19 patients. METHODS: Recombinant nucleocapsid protein and the S1, S2 and receptor binding domain (RBD) of the spike protein of SARS-CoV-2 were used as target antigens in the COVID-19 IBs. Specificity of the IB assay was established with 231 sera from persons with allergy, unrelated viral infections, autoimmune conditions and suspected tick-borne diseases, and 32 goat antisera to human influenza proteins. IgG and IgM COVID-19 IBs assays were performed on 84 sera obtained at different times after a positive RT-qPCR test from 37 COVID-19 patients with mild symptoms. RESULTS: Criteria for determining overall IgG and IgM antibody positivity using the four SARS-CoV-2 proteins were developed by optimizing specificity and sensitivity in the COVID-19 IgG and IgM IB assays. The estimated sensitivities and specificities of the COVID-19 IgG and IgM IBs for IgG and IgM antibodies individually or for either IgG or IgM antibodies meet the US recommendations for laboratory serological diagnostic tests. The proportion of IgM-positive sera from the COVID-19 patients following an RT-qPCR positive test was maximal at 83% before 10 days and decreased to 0% after 100 days, while the proportions of IgG-positive sera tended to plateau between days 11 and 65 at 78-100% and fall to 44% after 100 days. Detection of either IgG or IgM antibodies was better than IgG or IgM alone for assessing seroconversion in COVID-19. Both IgG and IgM antibodies detected RBD less frequently than S1, S2 and N proteins. CONCLUSIONS: The multiplex COVID-19 IB assays offer many advantages for simultaneously evaluating antibody responses to different SARS-CoV-2 proteins in COVID-19 patients.

Sex Matters: Male Hamsters Are More Susceptible to Lethal Infection with Lower Doses of Pathogenic Leptospira than Female Hamsters.

A somewhat contradictory published body of evidence suggests that sex impacts severity outcomes of human leptospirosis. In this study, we used an acute animal model of disease to analyze leptospirosis in male and female hamsters infected side by side with low but increasing doses of Leptospira interrogans serovar Copenhageni. We found that male hamsters were considerably more susceptible to leptospirosis, given that only 6.3% survived infection, whereas 68.7% of the females survived the same infection doses. In contrast to the females, male hamsters had high burdens of L. interrogans in kidney and high histopathological scores after exposure to low infection doses (∼103 bacteria). In hamsters infected with higher doses of L. interrogans (∼104 bacteria), differences in pathogen burdens as well as cytokine and fibrosis transcript levels in kidney were not distinct between sexes. Our results indicate that male hamsters infected with L. interrogans are more susceptible to severe leptospirosis after exposure to lower infectious doses than females.

Eyedrop Inoculation Causes Sublethal Leptospirosis in Mice.

Leptospirosis is potentially a fatal zoonosis acquired by contact of skin and mucosal surfaces with soil and water contaminated with infected urine. We analyzed the outcome of infection of C3H/HeJ mice with Leptospira interrogans serovar Copenhageni using an enzootic mode of transmission, the conjunctival route. Infection led to weight loss and L. interrogans dissemination from blood to urine, and spirochetes were detected in blood and urine simultaneously. The infectious dose that led to consistent dissemination to kidney after conjunctival infection was ∼108 leptospires. Interestingly, a lower number of spirochetes appeared to colonize the kidney, given that we quantified ∼105 and ∼10 leptospires per µl of urine and per µg of kidney, respectively. Leptospira-specific IgM and IgG were detected at 15 days postinfection, and isotyping of the Ig subclass showed that the total IgG response switched from an IgG1 response to an IgG3 response after infection with L. interrogans Histological periodic acid-Schiff D staining of infected kidney showed interstitial nephritis, mononuclear cell infiltrates, and reduced size of glomeruli. Quantification of proinflammatory immunomediators in kidney showed that keratinocyte-derived chemokine, macrophage inflammatory protein 2, RANTES, tumor necrosis factor alpha, gamma interferon, and interleukin-10 were upregulated in infected mice. We show that the kinetics of disease progression after infection via the ocular conjunctiva is delayed compared with infection via the standard intraperitoneal route. Differences may be related to the number of L. interrogans spirochetes that succeed in overcoming the natural defenses of the ocular conjunctiva and transit through tissue.

Mouse model for sublethal Leptospira interrogans infection.

Although Leptospira can infect a wide range of mammalian species, most studies have been conducted in golden Syrian hamsters, a species particularly sensitive to acute disease. Chronic disease has been well characterized in the rat, one of the natural reservoir hosts. Studies in another asymptomatic reservoir host, the mouse, have occasionally been done and have limited infection to mice younger than 6 weeks of age. We analyzed the outcome of sublethal infection of C3H/HeJ mice older than age 10 weeks with Leptospira interrogans serovar Copenhageni. Infection led to bloodstream dissemination of Leptospira, which was followed by urinary shedding, body weight loss, hypothermia, and colonization of the kidney by live spirochetes 2 weeks after infection. In addition, Leptospira dissemination triggered inflammation in the kidney but not in the liver or lung, as determined by increased levels of mRNA transcripts for the keratinocyte-derived chemokine, RANTES, macrophage inflammatory protein 2, tumor necrosis factor alpha, interleukin-1ß, inducible nitric oxide synthase, interleukin-6, and gamma interferon in kidney tissue. The acquired humoral response to Leptospira infection led to the production of IgG mainly of the IgG1 subtype. Flow cytometric analysis of splenocytes from infected mice revealed that cellular expansion was primarily due to an increase in the levels of CD4(+) and double-negative T cells (not CD8(+) cells) and that CD4(+) T cells acquired a CD44(high) CD62L(low) effector phenotype not accompanied by increases in memory T cells. A mouse model for sublethal Leptospira infection allows understanding of the bacterial and host factors that lead to immune evasion, which can result in acute or chronic disease or resistance to infection (protection).

Genetic variation at a Yin-Yang 1 response site regulates the transcription of cyclin-dependent kinase inhibitor p18INK4C transcript in lupus-prone mice.

We have previously shown that a novel -74 C-to-T mutation in the promoter of the cyclin-dependent kinase inhibitor p18(Ink4c) (p18) gene was associated with a reduced p18 expression in B cells from mice carrying the Sle2c1 lupus susceptibility locus. To determine the function of the -74 C/T single nucleotide polymorphism, we have characterized the proximal promoter of the mouse p18 gene. Functional analysis of the 5' flanking region by sequential deletions revealed crucial elements between -300 and +1, confirming the in silico prediction that the -74 T allele created a novel Yin-Yang 1 (YY-1) binding site adjacent to an existing one common to both alleles. Moreover, we found that YY-1, E2F1, and Sp-1 can synergistically enhance the activity of the p18 promoter. Mutational inactivation revealed that YY-1 binding regulates the p18 activity in an allele-dependent fashion. EMSAs with splenic B cell extracts directly demonstrated that YY-1 binds to the p18 promoter with differences between the C and the T alleles. We also determined in vivo by chromatin immunoprecipitation that the T allele resulted in increased YY-1 and decreased Nrf-2 binding to the p18 promoter as compared with the C allele in B cells. Thus, YY-1 is a direct regulator of p18 gene expression in an allele-dependent fashion that is consistent with the lupus-associated T allele, inducing a lower p18 transcriptional activity by increasing YY-1 binding. These results establish the p18 -74 C/T mutation as the leading causal variant for the B1a cell expansion that characterizes the NZB and NZM2410 lupus-prone strains.

Cyclin-dependent kinase inhibitor Cdkn2c deficiency promotes B1a cell expansion and autoimmunity in a mouse model of lupus.

The lupus-prone NZM2410 mice present an expanded B1a cell population that we have mapped to the Sle2c1 lupus susceptibility locus. The expression of Cdkn2c, a gene encoding for cyclin-dependent kinase inhibitor p18(Ink4c) and located within Sle2c1, is significantly lower in B6.Sle2c1 B cells than in B6 B cells. To test the hypothesis that the B1a cell expansion in B6.Sle2c1 mice was due to a defective p18 expression, we analyzed the B1a cell phenotypes of p18-deficient C57BL/6 mice. We found a dose-dependent negative correlation between the number of B1a cells and p18 expression in B cells, with p18-deficient mice showing an early expansion of the peritoneal B1a cell pool. p18 deficiency enhanced the homeostatic expansion of B1a cells but not of splenic conventional B cells, and the elevated number of B6.Sle2c1 B1a cells was normalized by cyclin D2 deficiency. These data demonstrated that p18 is a key regulator of the size of the B1a cell pool. B6.p18(-/-) mice produced significant amounts of anti-DNA IgM and IgG, indicating that p18 deficiency contributes to humoral autoimmunity. Finally, we have shown that Sle2c1 increases lpr-associated lymphadenopathy and T cell-mediated pathology. B6.p18(-/-).lpr mice showed a greater lymphadenopathy than B6.Sle2c1.lpr mice, but their renal pathology was intermediate between that of B6.lpr and B6.Sle2c1.lpr mice. This indicated that p18-deficiency synergizes, at least partially, with lpr-mediated pathology. These results show that Cdkn2c contributes to lupus susceptibility by regulating the size of the B1a cell compartment and hence their contribution to autoimmunity.

Pre-B cell leukemia homeobox 1 is associated with lupus susceptibility in mice and humans.

Sle1a.1 is part of the Sle1 susceptibility locus, which has the strongest association with lupus nephritis in the NZM2410 mouse model. In this study, we show that Sle1a.1 results in the production of activated and autoreactive CD4(+) T cells. Additionally, Sle1a.1 expression reduces the peripheral regulatory T cell pool, as well as induces a defective response of CD4(+) T cells to the retinoic acid expansion of TGF-ß-induced regulatory T cells. At the molecular level, Sle1a.1 corresponds to an increased expression of a novel splice isoform of Pbx1, Pbx1-d. Pbx1-d overexpression is sufficient to induce an activated/inflammatory phenotype in Jurkat T cells and to decrease their apoptotic response to retinoic acid. PBX1-d is expressed more frequently in the CD4(+) T cells from lupus patients than from healthy controls, and its presence correlates with an increased central memory T cell population. These findings indicate that Pbx1 is a novel lupus susceptibility gene that regulates T cell activation and tolerance.

P66 is a bacterial mimic of CD47 that binds the anti-phagocytic receptor SIRPα and facilitates macrophage evasion by Borrelia burgdorferi.

Innate immunity, the first line of defense against pathogens, relies on efficient elimination of invading agents by phagocytes. In the co-evolution of host and pathogen, pathogens developed mechanisms to dampen and evade phagocytic clearance. Here, we report that bacterial pathogens can evade clearance by macrophages through mimicry at the mammalian anti-phagocytic "don't eat me" signaling axis between CD47 (ligand) and SIRPα (receptor). We identified a protein, P66, on the surface of Borrelia burgdorferi that, like CD47, is necessary and sufficient to bind the macrophage receptor SIRPα. Expression of the gene encoding the protein is required for bacteria to bind SIRPα or a high-affinity CD47 reagent. Genetic deletion of p66 increases phagocytosis by macrophages. Blockade of P66 during infection promotes clearance of the bacteria. This study demonstrates that mimicry of the mammalian anti-phagocytic protein CD47 by B. burgdorferi inhibits macrophage-mediated bacterial clearance. Such a mechanism has broad implications for understanding of host-pathogen interactions and expands the function of the established innate immune checkpoint receptor SIRPα. Moreover, this report reveals P66 as a novel therapeutic target in the treatment of Lyme Disease.

Cyclin-dependent kinase inhibitor Cdkn2c regulates B cell homeostasis and function in the NZM2410-derived murine lupus susceptibility locus Sle2c1.

Sle2c1 is an NZM2410- and NZB-derived lupus susceptibility locus that induces an expansion of the B1a cell compartment. B1a cells have a repertoire enriched for autoreactivity, and an expansion of this B cell subset occurs in several mouse models of lupus. A combination of genetic mapping and candidate gene analysis presents Cdkn2c, a gene encoding for cyclin-dependent kinase inhibitor p18(INK4c) (p18), as the top candidate gene for inducing the Slec2c1-associated expansion of B1a cells. A novel single nucleotide polymorphism in the NZB allele of the Cdkn2c promoter is associated with a significantly reduced Cdkn2c expression in the splenic B cells and peritoneal cavity B1a cells from Sle2c1-carrying mice, which leads to a defective G1 cell cycle arrest in splenic B cells and increased proliferation of peritoneal cavity B1a cells. As the cell cycle is differentially regulated in B1a and B2 cells, these results suggest that Cdkn2c plays a critical role in B1a cell self-renewal and that its impaired expression leads to an accumulation of these cells with high autoreactive potential.

Repurposing Disulfiram (Tetraethylthiuram Disulfide) as a Potential Drug Candidate against Borrelia burgdorferi In Vitro and In Vivo.

Lyme disease caused by the Borrelia burgdorferi (Bb or B. burgdorferi) is the most common vector-borne, multi-systemic disease in the USA. Although most Lyme disease patients can be cured with a course of the first line of antibiotic treatment, some patients are intolerant to currently available antibiotics, necessitating the development of more effective therapeutics. We previously found several drugs, including disulfiram, that exhibited effective activity against B. burgdorferi. In the current study, we evaluated the potential of repurposing the FDA-approved drug, disulfiram for its borreliacidal activity. Our results indicate disulfiram has excellent borreliacidal activity against both the log and stationary phase B. burgdorferi sensu stricto B31 MI. Treatment of mice with disulfiram eliminated the B. burgdorferi sensu stricto B31 MI completely from the hearts and urinary bladder by day 28 post infection. Moreover, disulfiram-treated mice showed reduced expressions of inflammatory markers, and thus they were protected from histopathology and cardiac organ damage. Furthermore, disulfiram-treated mice showed significantly lower amounts of total antibody titers (IgM and IgG) at day 21 and total IgG2b at day 28 post infection. FACS analysis of lymph nodes revealed a decrease in the percentage of CD19+ B cells and an increase in total percentage of CD3+ T cells, CD3+ CD4+ T helpers, and naive and effector memory cells in disulfiram-treated mice. Together, our findings suggest that disulfiram has the potential to be repurposed as an effective antibiotic for treating Lyme disease.

Azlocillin can be the potential drug candidate against drug-tolerant Borrelia burgdorferi sensu stricto JLB31.

Lyme disease is one of most common vector-borne diseases, reporting more than 300,000 cases annually in the United States. Treating Lyme disease during its initial stages with traditional tetracycline antibiotics is effective. However, 10-20% of patients treated with antibiotic therapy still shows prolonged symptoms of fatigue, musculoskeletal pain, and perceived cognitive impairment. When these symptoms persists for more than 6 months to years after completing conventional antibiotics treatment are called post-treatment Lyme disease syndrome (PTLDS). Though the exact reason for the prolongation of post treatment symptoms are not known, the growing evidence from recent studies suggests it might be due to the existence of drug-tolerant persisters. In order to identify effective drug molecules that kill drug-tolerant borrelia we have tested two antibiotics, azlocillin and cefotaxime that were identified by us earlier. The in vitro efficacy studies of azlocillin and cefotaxime on drug-tolerant persisters were done by semisolid plating method. The results obtained were compared with one of the currently prescribed antibiotic doxycycline. We found that azlocillin completely kills late log phase and 7-10 days old stationary phase B. burgdorferi. Our results also demonstrate that azlocillin and cefotaxime can effectively kill in vitro doxycycline-tolerant B. burgdorferi. Moreover, the combination drug treatment of azlocillin and cefotaxime effectively killed doxycycline-tolerant B. burgdorferi. Furthermore, when tested in vivo, azlocillin has shown good efficacy against B. burgdorferi in mice model. These seminal findings strongly suggests that azlocillin can be effective in treating B. burgdorferi sensu stricto JLB31 infection and furthermore in depth research is necessary to evaluate its potential use for Lyme disease therapy.

In vitro and in vivo evaluation of cephalosporins for the treatment of Lyme disease.

BACKGROUND: Lyme disease accounts for >90% of all vector-borne disease cases in the United States and affect ~300,000 persons annually in North America. Though traditional tetracycline antibiotic therapy is generally prescribed for Lyme disease, still 10%-20% of patients treated with current antibiotic therapy still show lingering symptoms. METHODS: In order to identify new drugs, we have evaluated four cephalosporins as a therapeutic alternative to commonly used antibiotics for the treatment of Lyme disease by using microdilution techniques like minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). We have determined the MIC and MBC of four drugs for three Borrelia burgdorferi s.s strains namely CA8, JLB31 and NP40. The binding studies were performed using in silico analysis. RESULTS: The MIC order of the four drugs tested is cefoxitin (1.25 µM/mL) > cefamandole (2.5 µM/mL), > cefuroxime (5 µM/mL) > cefapirin (10 µM/mL). Among the drugs that are tested in this study using in vivo C3H/HeN mouse model, cefoxitin effectively kills B. burgdorferi. The in silico analysis revealed that all four cephalosporins studied binds effectively to B. burgdorferi proteins, SecA subunit penicillin-binding protein (PBP) and Outer surface protein E (OspE). CONCLUSION: Based on the data obtained, cefoxitin has shown high efficacy killing B. burgdorferi at concentration of 1.25 µM/mL. In addition to it, cefoxitin cleared B. burgdorferi infection in C3H/HeN mice model at 20 mg/kg.

Pre-treatment with Lactobacillus plantarum prevents severe pathogenesis in mice infected with Leptospira interrogans and may be associated with recruitment of myeloid cells.

Recent estimates on global morbidity and mortality caused by Leptospirosis point to one million cases and almost 60,000 deaths a year worldwide, especially in resource poor countries. We analyzed how a commensal probiotic immunomodulator, Lactobacillus plantarum, affects Leptospira interrogans pathogenesis in a murine model of sub-lethal leptospirosis. We found that repeated oral pre-treatment of mice with live L. plantarum restored body weight to normal levels in mice infected with L. interrogans. Pre-treatment did not prevent L. interrogans access to the kidney but it affected the inflammatory response and it reduced histopathological signs of disease. Analysis of the immune cell profiles in lymphoid tissues of mice pre-treated with L. plantarum showed increased numbers of B cells as well as naïve and memory CD4+ helper T cell populations in uninfected mice that shifted towards increased numbers of effector CD4+ helper T in infected mice. CD8+ cytotoxic T cell profiles in pre-treated uninfected and infected mice mirrored the switch observed for CD4+ except that CD8+ memory T cells were not affected. In addition, pre-treatment led to increased populations of monocytes in lymphoid tissues of uninfected mice and to increased populations of macrophages in the same tissues of infected mice. Immunohistochemistry of kidney sections of pre-treated infected mice showed an enrichment of neutrophils and macrophages and a reduction of total leucocytes and T cells. Our results suggest that complex myeloid and T cell responses orchestrate the deployment of monocytes and other cells from lymphoid tissue and the recruitment of neutrophils and macrophages to the kidney, and that, the presence of these cells in the target organ may be associated with reductions in pathogenesis observed in infected mice treated with L. plantarum.

Transcriptional activation of Mina by Sp1/3 factors.

Mina is an epigenetic gene regulatory protein known to function in multiple physiological and pathological contexts, including pulmonary inflammation, cell proliferation, cancer and immunity. We showed previously that the level of Mina gene expression is subject to natural genetic variation linked to 21 SNPs occurring in the Mina 5' region. In order to explore the mechanisms regulating Mina gene expression, we set out to molecularly characterize the Mina promoter in the region encompassing these SNPs. We used three kinds of assays--reporter, gel shift and chromatin immunoprecipitation--to analyze a 2 kb genomic fragment spanning the upstream and intron 1 regions flanking exon 1. Here we discovered a pair of Mina promoters (P1 and P2) and a P1-specific enhancer element (E1). Pharmacologic inhibition and siRNA knockdown experiments suggested that Sp1/3 transcription factors trigger Mina expression through additive activity targeted to a cluster of four Sp1/3 binding sites forming the P1 promoter. These results set the stage for comprehensive analysis of Mina gene regulation from the context of tissue specificity, the impact of inherited genetic variation and the nature of upstream signaling pathways.

FoxM1 regulates re-annealing of endothelial adherens junctions through transcriptional control of beta-catenin expression.

Repair of the injured vascular intima requires a series of coordinated events that mediate both endothelial regeneration and reannealing of adherens junctions (AJs) to form a restrictive endothelial barrier. The forkhead transcription factor FoxM1 is essential for endothelial proliferation after vascular injury. However, little is known about mechanisms by which FoxM1 regulates endothelial barrier reannealing. Here, using a mouse model with endothelial cell (EC)-restricted disruption of FoxM1 (FoxM1 CKO) and primary cultures of ECs with small interfering RNA (siRNA)-mediated knockdown of FoxM1, we demonstrate a novel requisite role of FoxM1 in mediating endothelial AJ barrier repair through the transcriptional control of beta-catenin. In the FoxM1 CKO lung vasculature, we observed persistent microvessel leakage characterized by impaired reannealing of endothelial AJs after endothelial injury. We also showed that FoxM1 directly regulated beta-catenin transcription and that reexpression of beta-catenin rescued the defective AJ barrier-reannealing phenotype of FoxM1-deficient ECs. Knockdown of beta-catenin mimicked the phenotype of defective barrier recovery seen in FoxM1-deficient ECs. These data demonstrate that FoxM1 is required for reannealing of endothelial AJs in order to form a restrictive endothelial barrier through transcriptional control of beta-catenin expression. Therefore, means of activating FoxM1-mediated endothelial repair represent a new therapeutic strategy for the treatment of inflammatory vascular diseases associated with persistent vascular barrier leakiness such as acute lung injury.

Persistent eNOS activation secondary to caveolin-1 deficiency induces pulmonary hypertension in mice and humans through PKG nitration.

Pulmonary hypertension (PH) is an unremitting disease defined by a progressive increase in pulmonary vascular resistance leading to right-sided heart failure. Using mice with genetic deletions of caveolin 1 (Cav1) and eNOS (Nos3), we demonstrate here that chronic eNOS activation secondary to loss of caveolin-1 can lead to PH. Consistent with a role for eNOS in the pathogenesis of PH, the pulmonary vascular remodeling and PH phenotype of Cav1-/- mice were absent in Cav1-/-Nos3-/- mice. Further, treatment of Cav1-/- mice with either MnTMPyP (a superoxide scavenger) or l-NAME (a NOS inhibitor) reversed their pulmonary vascular pathology and PH phenotype. Activation of eNOS in Cav1-/- lungs led to the impairment of PKG activity through tyrosine nitration. Moreover, the PH phenotype in Cav1-/- lungs could be rescued by overexpression of PKG-1. The clinical relevance of the data was indicated by the observation that lung tissue from patients with idiopathic pulmonary arterial hypertension demonstrated increased eNOS activation and PKG nitration and reduced caveolin-1 expression. Together, these data show that loss of caveolin-1 leads to hyperactive eNOS and subsequent tyrosine nitration-dependent impairment of PKG activity, which results in PH. Thus, targeting of PKG nitration represents a potential novel therapeutic strategy for the treatment of PH.