Early diagnosis of Ehrlichia ewingii infection in a lung transplant recipient by peripheral blood smear.

Ehrlichiosis in lung transplant (LT) recipients is associated with severe outcomes. Ehrlichia ewingii is a less frequent cause of symptomatic ehrlichiosis, characterized by cytoplasmic inclusions (morulae) within circulating neutrophils. We report a case of E. ewingii infection in an LT recipient diagnosed promptly by blood smear exam and confirmed with molecular studies.

High serum CXCL10 in Rickettsia conorii infection is endothelial cell mediated subsequent to whole blood activation.

BACKGROUND: The pathophysiological hallmark of Rickettsia conorii (R. conorii) infection comprises infection of endothelial cells with perivascular infiltration of T-cells and macrophages. Although interferon (IFN)-γ-induced protein 10 (IP-10)/CXCL10 is induced during vascular inflammation, data on CXCL10 in R. conorii infection is scarce. METHODS: Serum CXCL10 was analyzed in two cohorts of southern European patients with R. conorii infection using multiplex cytokine assays. The mechanism of R. conorii-induced CXCL10 release was examined ex vivo using human whole blood interacting with endothelial cells. RESULTS: (i) At admission, R. conorii infected patients had excessively increased CXCL10 levels, similar in the Italian (n=32, ∼56-fold increase vs controls) and the Spanish cohort (n=38, ∼68-fold increase vs controls), followed by a marked decrease after recovery. The massive CXCL10 increase was selective since it was not accompanied with similar changes in other cytokines. (ii) Heat-inactivated R. conorii induced a marked CXCL10 increase when whole blood and endothelial cells were co-cultured. Even plasma obtained from R. conorii-exposed whole blood induced a marked CXCL10 release from endothelial cells, comparable to the levels found in serum of R. conorii-infected patients. Bacteria alone did not induce CXCL10 production in endothelial cells, macrophages or smooth muscle cells. CONCLUSIONS: We show a massive and selective serum CXCL10 response in R. conorii-infected patients, likely reflecting release from infected endothelial cells characterized by infiltrating T cells and monocytes. The CXCL10 response could contribute to T-cell infiltration within the infected organ, but the pathologic consequences of CXCL10 in clinical R. conorii infection remain to be defined.

Increased expression of the homeostatic chemokines CCL19 and CCL21 in clinical and experimental Rickettsia conorii infection.

BACKGROUND: Based on their essential role in concerting immunological and inflammatory responses we hypothesized that the homeostatic chemokines CCL19 and CCL21 may play a pathogenic role in rickettsiae infection. METHODS: Serum levels of CCL19 and CCL21 in patients with R. africae and R. conorii infection were analyzed by enzyme immunoassays. Lungs from R. conorii infected mice were examined for CCL19, CCL21 and CCR7 expression by immunohistochemistry. RESULTS: We found that patients with R. africae infection (n = 15) and in particular those with R. conorii infection (n = 16) had elevated serum levels of CCL19 on admission, with a decline during follow-up. While a similar pattern was seen for CCL21 in R. africae infection, patients with R. conorii infection showed persistently increased CCL21 levels during follow-up. In experimental R. conorii infection, we found strong immunostaining of CCL19 and CCL21 in the lungs, particularly in individuals that had received lethal doses. Immunofluorescence showed co-localization of CCR7 to endothelial cells, macrophages and fibroblasts within the lung tissue of R. conorii infected mice. CONCLUSIONS: Our findings suggest that the CCL19/CCL21/CCR7 axis is up-regulated during R. africae and in particular during R. conorii infection, which may potentially contribute to the pathogenesis of these disorders.

A Bacteriophage-Based, Highly Efficacious, Needle- and Adjuvant-Free, Mucosal COVID-19 Vaccine.

The U.S. Food and Drug Administration-authorized mRNA- and adenovirus-based SARS-CoV-2 vaccines are intramuscularly injected in two doses and effective in preventing COVID-19, but they do not induce efficient mucosal immunity or prevent viral transmission. Here, we report the first noninfectious, bacteriophage T4-based, multicomponent, needle- and adjuvant-free, mucosal vaccine harboring engineered Spike trimers on capsid exterior and nucleocapsid protein in the interior. Intranasal administration of two doses of this T4 SARS-CoV-2 vaccine 21 days apart induced robust mucosal immunity, in addition to strong systemic humoral and cellular immune responses. The intranasal vaccine induced broad virus neutralization antibody titers against multiple variants, Th1-biased cytokine responses, strong CD4+ and CD8+ T cell immunity, and high secretory IgA titers in sera and bronchoalveolar lavage specimens from vaccinated mice. All of these responses were much stronger in intranasally vaccinated mice than those induced by the injected vaccine. Furthermore, the nasal vaccine provided complete protection and sterilizing immunity against the mouse-adapted SARS-CoV-2 MA10 strain, the ancestral WA-1/2020 strain, and the most lethal Delta variant in both BALB/c and human angiotensin converting enzyme (hACE2) knock-in transgenic mouse models. In addition, the vaccine elicited virus-neutralizing antibodies against SARS-CoV-2 variants in bronchoalveolar lavage specimens, did not affect the gut microbiota, exhibited minimal lung lesions in vaccinated and challenged mice, and is completely stable at ambient temperature. This modular, needle-free, phage T4 mucosal vaccine delivery platform is therefore an excellent candidate for designing efficacious mucosal vaccines against other respiratory infections and for emergency preparedness against emerging epidemic and pandemic pathogens. IMPORTANCE According to the World Health Organization, COVID-19 may have caused ~15-million deaths across the globe and is still ravaging the world. Another wave of ~100 million infections is predicted in the United States due to the emergence of highly transmissible immune-escaped Omicron variants. The authorized vaccines would not prevent these transmissions since they do not trigger mucosal immunity. We circumvented this limitation by developing a needle-free, bacteriophage T4-based, mucosal vaccine. This intranasally administered vaccine generates superior mucosal immunity in mice, in addition to inducing robust humoral and cell-mediated immune responses, and provides complete protection and sterilizing immunity against SARS-CoV-2 variants. The vaccine is stable, adjuvant-free, and cost-effectively manufactured and distributed, making it a strategically important next-generation COVID vaccine for ending this pandemic.

Surface-expressed enolase contributes to the pathogenesis of clinical isolate SSU of Aeromonas hydrophila.

In this study, we demonstrated that the surface-expressed enolase from diarrheal isolate SSU of Aeromonas hydrophila bound to human plasminogen and facilitated the latter's tissue-type plasminogen activator-mediated activation to plasmin. The bacterial surface-bound plasmin was more resistant to the action of its specific physiological inhibitor, the antiprotease alpha(2)-antiplasmin. We found that immunization of mice with purified recombinant enolase significantly protected the animals against a lethal challenge dose of wild-type (WT) A. hydrophila. Minimal histological changes were noted in organs from mice immunized with enolase and then challenged with WT bacteria compared to severe pathological changes found in the infected and nonimmunized group of animals. This correlated with the smaller bacterial load of WT bacteria in the livers and spleens of enolase-immunized mice than that found in the nonimmunized controls. We also showed that the enolase gene could potentially be important for the viability of A. hydrophila SSU as we could delete the chromosomal copy of the enolase gene only when another copy of the targeted gene was supplied in trans. By site-directed mutagenesis, we altered five lysine residues located at positions 343, 394, 420, 427, and 430 of enolase in A. hydrophila SSU; the mutated forms of enolase were hyperexpressed in Escherichia coli, and the proteins were purified. Our results indicated that lysine residues at positions 420 and 427 of enolase were crucial in plasminogen-binding activity. We also identified a stretch of amino acid residues ((252)FYDAEKKEY(260)) in the A. hydrophila SSU enolase involved in plasminogen binding. To our knowledge, this is the first report of the direct involvement of surface-expressed enolase in the pathogenesis of A. hydrophila SSU infections and of any gram-negative bacteria in general.

David Versus Goliath: The Clinical Consequences of an Influenza A Viral Infection in an Unvaccinated Patient.

Acute influenza virus (AIV) infection can manifest as a severe life-threating illness in patients who are not vaccinated, and furthermore, have comorbidities that place them at risk for rapid respiratory decompensation. Each year influenza causes death in individuals with high risk for contracting this infection, although the illness is preventable by vaccination. Complications of AIV infection, such as bacterial pneumonia are treatable, but other severe complications such as acute respiratory distress syndrome (ARDS) leading to diffuse alveolar damage (DAD) are limited to supportive therapy and self-resolution. In most cases, ARDS leading to DAD is fatal, due to the insidious severity of symptoms which lead to rapid oxygen desaturation without correction, and despite supportive therapy. Regardless of a poor prognosis, the clinical signs and symptoms are congruent with imaging and attest to the importance of vaccination, which protect against high mortality rates.

The absence of Toll-like receptor 4 signaling in C3H/HeJ mice predisposes them to overwhelming rickettsial infection and decreased protective Th1 responses.

The importance of toll-like receptor 4 (TLR4) in immunity to rickettsiae remains elusive. To investigate the role of TLR4 in protection against rickettsioses, we utilized C3H/HeJ mice, which are naturally defective in TLR4 signaling, and compared the responses of C3H/HeN and C3H/HeJ mice following intravenous inoculation with Rickettsia conorii. Mice genetically defective in TLR4 signaling developed overwhelming, fatal rickettsial infections when given an inoculum that was nonfatal for TLR4-competent mice. In addition, mice lacking the ability to signal through TLR4 had significantly greater rickettsial burdens in vivo. Moreover, we observed greater concentrations of the cytokines interleukin 6 (IL-6), tumor necrosis factor alpha, IL-12p40, IL-12p70, and IL-17 in the sera of mice with intact TLR4 function as well as significantly greater quantities of activated CD4(+) and CD8(+) T lymphocytes. Additionally, we also observed that Th17 cells were present only in TLR4-competent mice, suggesting an important role for TLR4 ligation in the activation of this subset. In agreement with these data, we also observed significantly greater percentages of immunosuppressive regulatory T cells in the spleen during infection in TLR4-defective mice. Together, these data demonstrate that, while rickettsiae do not contain endotoxic lipopolysaccharide, they nevertheless initiate TLR4-specific immune responses, and these responses are important in protection.

Braun lipoprotein (Lpp) contributes to virulence of yersiniae: potential role of Lpp in inducing bubonic and pneumonic plague.

Yersinia pestis evolved from Y. pseudotuberculosis to become the causative agent of bubonic and pneumonic plague. We identified a homolog of the Salmonella enterica serovar Typhimurium lipoprotein (lpp) gene in Yersinia species and prepared lpp gene deletion mutants of Y. pseudotuberculosis YPIII, Y. pestis KIM/D27 (pigmentation locus minus), and Y. pestis CO92 with reduced virulence. Mice injected via the intraperitoneal route with 5 x 10(7) CFU of the Deltalpp KIM/D27 mutant survived a month, even though this would have constituted a lethal dose for the parental KIM/D27 strain. Subsequently, these Deltalpp KIM/D27-injected mice were solidly protected against an intranasally administered, highly virulent Y. pestis CO92 strain when it was given as five 50% lethal doses (LD(50)). In a parallel study with the pneumonic plague mouse model, after 72 h postinfection, the lungs of animals infected with wild-type (WT) Y. pestis CO92 and given a subinhibitory dose of levofloxacin had acute inflammation, edema, and masses of bacteria, while the lung tissue appeared essentially normal in mice inoculated with the Deltalpp mutant of CO92 and given the same dose of levofloxacin. Importantly, while WT Y. pestis CO92 could be detected in the bloodstreams and spleens of infected mice at 72 h postinfection, the Deltalpp mutant of CO92 could not be detected in those organs. Furthermore, the levels of cytokines/chemokines detected in the sera were significantly lower in animals infected with the Deltalpp mutant than in those infected with WT CO92. Additionally, the Deltalpp mutant was more rapidly killed by macrophages than was the WT CO92 strain. These data provided evidence that the Deltalpp mutants of yersiniae were significantly attenuated and could be useful tools in the development of new vaccines.

Emerging pathogens: challenges and successes of molecular diagnostics.

More than 50 emerging and reemerging pathogens have been identified during the last 40 years. Until 1992 when the Institute of Medicine issued a report that defined emerging infectious diseases, medicine had been complacent about such infectious diseases despite the alarm bells of infections with human immunodeficiency virus. Molecular tools have proven useful in discovering and characterizing emerging viruses and bacteria such as Sin Nombre virus (hantaviral pulmonary syndrome), hepatitis C virus, Bartonella henselae (cat scratch disease, bacillary angiomatosis), and Anaplasma phagocytophilum (human granulocytotropic anaplasmosis). The feasibility of applying molecular diagnostics to dangerous, fastidious, and uncultivated agents for which conventional tests do not yield timely diagnoses has achieved proof of concept for many agents, but widespread use of cost-effective, validated commercial assays has yet to occur. This review presents representative emerging viral respiratory infections, hemorrhagic fevers, and hepatitides, as well as bacterial and parasitic zoonotic, gastrointestinal, and pulmonary infections. Agent characteristics, epidemiology, clinical manifestations, and diagnostic methods are tabulated for another 22 emerging viruses and five emerging bacteria. The ongoing challenge to the field of molecular diagnostics is to apply contemporary knowledge to facilitate agent diagnosis as well as to further discoveries of novel pathogens.