Performance of the Xpert™ Mpox PCR assay with oropharyngeal, anorectal, and cutaneous lesion swab specimens.

Anorectal and oropharyngeal exposures are implicated in sexual transmission of mpox, but authorized assays in the United States are only validated with cutaneous lesion swabs. Diagnostic assays for anorectal and oropharyngeal swabs are needed to address potential future outbreaks. The Cepheid Xpert® Mpox is the first point-of-care assay to receive FDA emergency use authorization in the United States and would be a valuable tool for evaluating these sample types. Our exploratory study demonstrates 100 % positive agreement with our in-house PCR assay for natural positive anorectal and oropharyngeal specimens and 92 % sensitivity with low-positive spiked specimens. The Xpert® assay detected viral DNA in specimens not detected by our reference PCR assay from four participants with mpox DNA at other sites, suggesting it may be more sensitive at low viral loads. In conclusion, the validation of the Xpert® for oropharyngeal and anorectal sample types can be rapidly achieved if clinical need returns and prospective samples become available.

Multisite Mpox Infection and Viral Dynamics Among Persons With HIV in Metro Atlanta.

The 2022 mpox outbreak primarily involved sexual transmission among men who have sex with men and disproportionately affected persons with human immunodeficiency virus (HIV). We examined viral dynamics and clinical features in a cohort evaluated for mpox infection at a comprehensive HIV clinic in Atlanta, Georgia. Viral DNA was found in 8 oropharyngeal and 5 anorectal specimens among 10 mpox cases confirmed by lesion swab polymerase chain reaction. Within-participant anatomic site of lowest cycle threshold (Ct) value varied, and lower Ct values were found in oropharyngeal and anorectal swabs when corresponding symptoms were present. This provides insight into mpox infection across multiple anatomic sites among people with HIV.

Viral and host factors associated with SARS-CoV-2 disease severity in Georgia, USA.

While SARS-CoV-2 vaccines have shown strong efficacy, their suboptimal uptake combined with the continued emergence of new viral variants raises concerns about the ongoing and future public health impact of COVID-19. We investigated viral and host factors, including vaccination status, that were associated with SARS-CoV-2 disease severity in a setting with low vaccination rates. We analyzed clinical and demographic data from 1,957 individuals in the state of Georgia, USA, coupled with viral genome sequencing from 1,185 samples. We found no difference in disease severity between individuals infected with Delta and Omicron variants among the participants in this study, after controlling for other factors, and we found no specific mutations associated with disease severity. Compared to those who were unvaccinated, vaccinated individuals experienced less severe SARS-CoV-2 disease, and the effect was similar for both variants. Vaccination within 270 days before infection was associated with decreased odds of moderate and severe outcomes, with the strongest association observed at 91-270 days post-vaccination. Older age and underlying health conditions, especially immunosuppression and renal disease, were associated with increased disease severity. Overall, this study provides insights into the impact of vaccination status, variants/mutations, and clinical factors on disease severity in SARS-CoV-2 infection when vaccination rates are low. Understanding these associations will help refine and reinforce messaging around the crucial importance of vaccination in mitigating the severity of SARS-CoV-2 disease.

Sensitivity of rapid antigen tests against SARS-CoV-2 Omicron and Delta variants.

Rapid antigen tests (RATs) have become an invaluable tool for combating the COVID-19 pandemic. However, concerns have been raised regarding the ability of existing RATs to effectively detect emerging SARS-CoV-2 variants. We compared the performance of 10 commercially available, emergency use authorized RATs against the Delta and Omicron SARS-CoV-2 variants using both individual patient and serially diluted pooled clinical samples. The RATs exhibited lower sensitivity for Omicron samples when using PCR cycle threshold (CT) value (a rough proxy for RNA concentration) as the comparator. Interestingly, however, they exhibited similar sensitivity for Omicron and Delta samples when using quantitative antigen concentration as the comparator. We further found that the Omicron samples had lower ratios of antigen to RNA, which offers a potential explanation for the apparent lower sensitivity of RATs for that variant when using C T value as a reference. Our findings underscore the complexity in assessing RAT performance against emerging variants and highlight the need for ongoing evaluation in the face of changing population immunity and virus evolution.

Investigation of Blood Plasma Viral Nucleocapsid Antigen as a Marker of Active Severe Acute Respiratory Syndrome Coronavirus 2 Omicron Variant Infection.

Background: Nasopharyngeal qualitative reverse-transcription polymerase chain reaction (RT-PCR) is the gold standard for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but it is not practical or sufficient in every clinical scenario due to its inability to distinguish active from resolved infection. Alternative or adjunct testing may be needed to guide isolation precautions and treatment in patients admitted to the hospital. Methods: We performed a single-center, retrospective analysis of residual clinical specimens and medical record data to examine blood plasma nucleocapsid antigen as a candidate biomarker of active SARS-CoV-2. Adult patients admitted to the hospital or presenting to the emergency department with SARS-CoV-2 ribonucleic acid (RNA) detected by RT-PCR from a nasopharyngeal swab specimen were included. Both nasopharyngeal swab and a paired whole blood sample were required to be available for analysis. Results: Fifty-four patients were included. Eight patients had positive nasopharyngeal swab virus cultures, 7 of whom (87.5%) had concurrent antigenemia. Nineteen (79.2%) of 24 patients with detectable subgenomic RNA and 20 (80.0%) of 25 patients with N2 RT-PCR cycle threshold ≤ 33 had antigenemia. Conclusions: Most individuals with active SARS-CoV-2 infection are likely to have concurrent antigenemia, but there may be some individuals with active infection in whom antigenemia is not detectable. The potential for high sensitivity and convenience of a blood test prompts interest in further investigation as a screening tool to reduce reliance on nasopharyngeal swab sampling and as an adjunct diagnostic test to aid in clinical decision making during the period after acute coronavirus disease 2019.

Sensitivity of Rapid Antigen Tests Against SARS-CoV-2 Omicron and Delta Variants.

Rapid Antigen Tests (RAT) have become an invaluable tool for combating the COVID-19 pandemic. However, concerns have been raised regarding the ability of existing RATs to effectively detect emerging SARS-CoV-2 variants. We compared the performance of eight commercially available, emergency use authorized RATs against the Delta and Omicron SARS-CoV-2 variants using individual patient and serially diluted pooled clinical samples. The RATs exhibited lower sensitivity for Omicron samples when using PCR Cycle threshold (C T ) value (a proxy for RNA concentration) as the comparator. Interestingly, however, they exhibited similar sensitivity for Omicron and Delta samples when using quantitative antigen concentration as the comparator. We further found that the Omicron samples had lower ratios of antigen to RNA, which offers a potential explanation for the apparent lower sensitivity of RATs for that variant when using C T value as a reference. Our findings underscore the complexity in assessing RAT performance against emerging variants and highlight the need for ongoing evaluation in the face of changing population immunity and virus evolution.

Nucleocapsid Antigenemia in Patients Receiving Anti-CD20 Therapy With Protracted COVID-19.

Immunocompromised patients with prolonged coronavirus disease 2019 symptoms present diagnostic and therapeutic challenges. We measured viral nucleocapsid antigenemia in 3 patients treated with anti-CD20 immunotherapy who acquired severe acute respiratory syndrome coronavirus 2 infection and experienced protracted symptoms. Our results support nucleocapsid antigenemia as a marker of persistent infection and therapeutic response.

Efficacy of therapeutic plasma exchange in reducing the incidence of recurrent pancreatitis related to familial chylomicronemia.

BACKGROUND: Familial chylomicronemia syndrome (FCS) is caused by a genetic defect in triglyceride (TG) metabolism that leads to severe hypertriglyceridemia, which in turn is associated with multiple morbidities and may cause severe pancreatitis that is both recurrent and progressive. Management of hypertriglyceridemia in FCS is challenging, as both dietary and medical interventions are often ineffective. Therapeutic plasma exchange (TPE) has been shown to rapidly decrease circulating levels of chylomicrons and TGs in patients presenting with acute hypertriglyceridemic-associated pancreatitis. Conversely, limited evidence exists to suggest that prophylactic use of TPE is effective at preventing recurrence of acute pancreatitis. CASE REPORT: Herein, we report our experience with the use of chronic, prophylactic TPE to reduce the incidence of recurrent acute pancreatitis in a patient with FCS.

Prolonged partial thromboplastin time: To mix or not to mix - is that the question?

Routine mixing studies are frequently used to evaluate patients presenting with prolonged partial thromboplastin times (PTT) and/or prothrombin times (PT). Unfortunately, mixing studies have a number of inherent limitations including lack of standardization in terms of what defines normal pooled plasma (NPP), the processing of a patient's plasma for platelet removal (platelet poor plasma versus platelet-free plasma), performance of appropriate controls, conducting an incubation step to evaluate for a time and temperature dependent inhibitor, and finally interpretation of test results. Moreover, misinterpretation of study results can lead to a delayed or incorrect diagnosis or worse, inappropriate treatment. Within this manuscript, we present four cases illustrating the shortcomings associated with inappropriate utilization and interpretation of routine mixing studies; and present practical steps for managing abnormal PT or PTT results.

Combating Multidrug-Resistant Pathogens with Host-Directed Nonantibiotic Therapeutics.

Earlier, we reported that three Food and Drug Administration-approved drugs, trifluoperazine (TFP; an antipsychotic), amoxapine (AXPN; an antidepressant), and doxapram (DXP; a breathing stimulant), identified from an in vitro murine macrophage cytotoxicity screen, provided mice with 40 to 60% protection against pneumonic plague when administered at the time of infection for 1 to 3 days. In the present study, the therapeutic potential of these drugs against pneumonic plague in mice was further evaluated when they were administered at up to 48 h postinfection. While the efficacy of TFP was somewhat diminished as treatment was delayed to 24 h, the protection of mice with AXPN and DXP increased as treatment was progressively delayed to 24 h. At 48 h postinfection, these drugs provided the animals with significant protection (up to 100%) against challenge with the agent of pneumonic or bubonic plague when they were administered in combination with levofloxacin. Likewise, when they were used in combination with vancomycin, all three drugs provided mice with 80 to 100% protection from fatal oral Clostridium difficile infection when they were administered at 24 h postinfection. Furthermore, AXPN provided 40 to 60% protection against respiratory infection with Klebsiella pneumoniae when it was administered at the time of infection or at 24 h postinfection. Using the same in vitro cytotoxicity assay, we identified an additional 76/780 nonantibiotic drugs effective against K. pneumoniae For Acinetobacter baumannii, 121 nonantibiotic drugs were identified to inhibit bacterium-induced cytotoxicity in murine macrophages. Of these 121 drugs, 13 inhibited the macrophage cytotoxicity induced by two additional multiple-antibiotic-resistant strains. Six of these drugs decreased the intracellular survival of all three A. baumannii strains in macrophages. These results provided further evidence of the broad applicability and utilization of drug repurposing screening to identify new therapeutics to combat multidrug-resistant pathogens of public health concern.

Identification of New Virulence Factors and Vaccine Candidates for Yersinia pestis.

Earlier, we reported the identification of new virulence factors/mechanisms of Yersinia pestis using an in vivo signature-tagged mutagenesis (STM) screening approach. From this screen, the role of rbsA, which encodes an ATP-binding protein of ribose transport system, and vasK, an essential component of the type VI secretion system (T6SS), were evaluated in mouse models of plague and confirmed to be important during Y. pestis infection. However, many of the identified genes from the screen remained uncharacterized. In this study, in-frame deletion mutants of ypo0815, ypo2884, ypo3614-3168 (cyoABCDE), and ypo1119-1120, identified from the STM screen, were generated. While ypo0815 codes for a general secretion pathway protein E (GspE) of the T2SS, the ypo2884-encoded protein has homology to the ßγ crystallin superfamily, cyoABCDE codes for the cytochrome o oxidase operon, and the ypo1119-1120 genes are within the Tol-Pal system which has multiple functions. Additionally, as our STM screen identified three T6SS-associated genes, and, based on in silico analysis, six T6SS clusters and multiple homologs of the T6SS effector hemolysin-coregulated protein (Hcp) exist in Y. pestis CO92, we also targeted these T6SS clusters and effectors for generating deletion mutants. These deletion mutant strains exhibited varying levels of attenuation (up to 100%), in bubonic or pneumonic murine infection models. The attenuation could be further augmented by generation of combinatorial deletion mutants, namely ΔlppΔypo0815, ΔlppΔypo2884, ΔlppΔcyoABCDE, ΔvasKΔhcp6, and Δypo2720-2733Δhcp3. We earlier showed that deletion of the lpp gene, which encodes Braun lipoprotein (Lpp) and activates Toll-like receptor-2, reduced virulence of Y. pestis CO92 in murine models of bubonic and pneumonic plague. The surviving mice infected with ΔlppΔcyoABCDE, ΔvasKΔhcp6, and Δypo2720-2733Δhcp3 mutant strains were 55-100% protected upon subsequent re-challenge with wild-type CO92 in a pneumonic model. Further, evaluation of the attenuated T6SS mutant strains in vitro revealed significant alterations in phagocytosis, intracellular survival in murine macrophages, and their ability to induce cytotoxic effects on macrophages. The results reported here provide further evidence of the utility of the STM screening approach for the identification of novel virulence factors and to possibly target such genes for the development of novel live-attenuated vaccine candidates for plague.

A Bivalent Anthrax-Plague Vaccine That Can Protect against Two Tier-1 Bioterror Pathogens, Bacillus anthracis and Yersinia pestis.

Bioterrorism remains as one of the biggest challenges to global security and public health. Since the deadly anthrax attacks of 2001 in the United States, Bacillus anthracis and Yersinia pestis, the causative agents of anthrax and plague, respectively, gained notoriety and were listed by the CDC as Tier-1 biothreat agents. Currently, there is no Food and Drug Administration-approved vaccine against either of these threats for mass vaccination to protect general public, let alone a bivalent vaccine. Here, we report the development of a single recombinant vaccine, a triple antigen consisting of all three target antigens, F1 and V from Y. pestis and PA from B. anthracis, in a structurally stable context. Properly folded and soluble, the triple antigen retained the functional and immunogenicity properties of all three antigens. Remarkably, two doses of this immunogen adjuvanted with Alhydrogel® elicited robust antibody responses in mice, rats, and rabbits and conferred complete protection against inhalational anthrax and pneumonic plague. No significant antigenic interference was observed. Furthermore, we report, for the first time, complete protection of animals against simultaneous challenge with Y. pestis and the lethal toxin of B. anthracis, demonstrating that a single biodefense vaccine can protect against a bioterror attack with weaponized B. anthracis and/or Y. pestis. This bivalent anthrax-plague vaccine is, therefore, a strong candidate for stockpiling, after demonstration of its safety and immunogenicity in human clinical trials, as part of national preparedness against two of the deadliest bioterror threats.

Phospholipase A2-activating protein is associated with a novel form of leukoencephalopathy.

Leukoencephalopathies are a group of white matter disorders related to abnormal formation, maintenance, and turnover of myelin in the central nervous system. These disorders of the brain are categorized according to neuroradiological and pathophysiological criteria. Herein, we have identified a unique form of leukoencephalopathy in seven patients presenting at ages 2 to 4 months with progressive microcephaly, spastic quadriparesis, and global developmental delay. Clinical, metabolic, and imaging characterization of seven patients followed by homozygosity mapping and linkage analysis were performed. Next generation sequencing, bioinformatics, and segregation analyses followed, to determine a loss of function sequence variation in the phospholipase A2-activating protein encoding gene (PLAA). Expression and functional studies of the encoded protein were performed and included measurement of prostaglandin E2 and cytosolic phospholipase A2 activity in membrane fractions of fibroblasts derived from patients and healthy controls. Plaa-null mice were generated and prostaglandin E2 levels were measured in different tissues. The novel phenotype of our patients segregated with a homozygous loss-of-function sequence variant, causing the substitution of leucine at position 752 to phenylalanine, in PLAA, which causes disruption of the protein's ability to induce prostaglandin E2 and cytosolic phospholipase A2 synthesis in patients' fibroblasts. Plaa-null mice were perinatal lethal with reduced brain levels of prostaglandin E2 The non-functional phospholipase A2-activating protein and the associated neurological phenotype, reported herein for the first time, join other complex phospholipid defects that cause leukoencephalopathies in humans, emphasizing the importance of this axis in white matter development and maintenance.

New Insights into Autoinducer-2 Signaling as a Virulence Regulator in a Mouse Model of Pneumonic Plague.

The Enterobacteriaceae family members, including the infamous Yersinia pestis, the causative agent of plague, have a highly conserved interbacterial signaling system that is mediated by the autoinducer-2 (AI-2) quorum-sensing molecule. The AI-2 system is implicated in regulating various bacterial virulence genes in diverse environmental niches. Deletion of the gene encoding the synthetic enzyme for the AI-2 substrate, luxS, leads to either no significant change or, paradoxically, an increase in in vivo bacterial virulence. We showed that deletion of the rbsA and lsrA genes, components of ABC transport systems that interact with AI-2, synergistically disrupted AI-2 signaling patterns and resulted in a more-than-50-fold decrease in Y. pestis strain CO92 virulence in a stringent pneumonic plague mouse model. Deletion of luxS or lsrK (encoding AI-2 kinase) from the ΔrbsA ΔlsrA background strain or complementation of the ΔrbsA ΔlsrA mutant with the corresponding gene(s) reverted the virulence phenotype to that of the wild-type Y. pestis CO92. Furthermore, the administration of synthetic AI-2 in mice infected with the ΔrbsA ΔlsrA ΔluxS mutant strain attenuated this triple mutant to a virulence phenotype similar to that of the ΔrbsA ΔlsrA strain in a pneumonic plague model. Conversely, the administration of AI-2 to mice infected with the ΔrbsA ΔlsrA ΔluxS ΔlsrK mutant did not rescue animals from lethality, indicating the importance of the AI-2-LsrK axis in regulating bacterial virulence. By performing high-throughput RNA sequencing, the potential role of some AI-2-signaling-regulated genes that modulated bacterial virulence was determined. We anticipate that the characterization of AI-2 signaling in Y. pestis will lead to reexamination of AI-2 systems in other pathogens and that AI-2 signaling may represent a broad-spectrum therapeutic target to combat antibiotic-resistant bacteria, which represent a global crisis of the 21st century. IMPORTANCEYersinia pestis is the bacterial agent that causes the highly fatal disease plague. The organism represents a significant concern because of its potential use as a bioterror agent, beyond the several thousand naturally occurring human infection cases occurring globally each year. While there has been development of effective antibiotics, the narrow therapeutic window and challenges posed by the existence of antibiotic-resistant strains represent serious concerns. We sought to identify novel virulence factors that could potentially be incorporated into an attenuated vaccine platform or be targeted by novel therapeutics. We show here that a highly conserved quorum-sensing system, autoinducer-2, significantly affected the virulence of Y. pestis in a mouse model of pneumonic plague. We also identified steps in autoinducer-2 signaling which had confounded previous studies and demonstrated the potential for intervention in the virulence mechanism(s) of autoinducer-2. Our findings may have an impact on bacterial pathogenesis research in many other organisms and could result in identifying potential broad-spectrum therapeutic targets to combat antibiotic-resistant bacteria, which represent a global crisis of the 21st century.

Protective Immunity Elicited by Oral Immunization of Mice with Salmonella enterica Serovar Typhimurium Braun Lipoprotein (Lpp) and Acetyltransferase (MsbB) Mutants.

We evaluated the extent of attenuation and immunogenicity of the ΔlppAB and ΔlppAB ΔmsbB mutants of Salmonella enterica serovar Typhimurium when delivered to mice by the oral route. These mutants were deleted either for the Braun lipoprotein genes (lppA and lppB) or in combination with the msbB gene, which encodes an acetyltransferase required for lipid A modification of lipopolysaccharide. Both the mutants were attenuated (100% animal survival) and triggered robust innate and adaptive immune responses. Comparable levels of IgG and its isotypes were produced in mice infected with wild-type (WT) S. typhimurium or its aforementioned mutant strains. The ΔlppAB ΔmsbB mutant-immunized animals resulted in the production of higher levels of fecal IgA and serum cytokines during later stages of vaccination (adaptive response). A significant production of interleukin-6 from T-cells was also noted in the ΔlppAB ΔmsbB mutant-immunized mice when compared to that of the ΔlppAB mutant. On the other hand, IL-17A production was significantly more in the serum of ΔlppAB mutant-immunized mice (innate response) with a stronger splenic T-cell proliferative and tumor-necrosis factor-α production. Based on 2-dimensional gel analysis, alterations in the levels of several proteins were observed in both the mutant strains when compared to that in WT S. typhimurium and could be associated with the higher immunogenicity of the mutants. Finally, both ΔlppAB and ΔlppAB ΔmsbB mutants provided complete protection to immunized mice against a lethal oral challenge dose of WT S. typhimurium. Thus, these mutants may serve as excellent vaccine candidates and also provide a platform for delivering heterologous antigens.

A Replication-Defective Human Type 5 Adenovirus-Based Trivalent Vaccine Confers Complete Protection against Plague in Mice and Nonhuman Primates.

Currently, no plague vaccine exists in the United States for human use. The capsular antigen (Caf1 or F1) and two type 3 secretion system (T3SS) components, the low-calcium-response V antigen (LcrV) and the needle protein YscF, represent protective antigens of Yersinia pestis We used a replication-defective human type 5 adenovirus (Ad5) vector and constructed recombinant monovalent and trivalent vaccines (rAd5-LcrV and rAd5-YFV) that expressed either the codon-optimized lcrV or the fusion gene designated YFV (consisting of ycsF, caf1, and lcrV). Immunization of mice with the trivalent rAd5-YFV vaccine by either the intramuscular (i.m.) or the intranasal (i.n.) route provided protection superior to that with the monovalent rAd5-LcrV vaccine against bubonic and pneumonic plague when animals were challenged with Y. pestis CO92. Preexisting adenoviral immunity did not diminish the protective response, and the protection was always higher when mice were administered one i.n. dose of the trivalent vaccine (priming) followed by a single i.m. booster dose of the purified YFV antigen. Immunization of cynomolgus macaques with the trivalent rAd5-YFV vaccine by the prime-boost strategy provided 100% protection against a stringent aerosol challenge dose of CO92 to animals that had preexisting adenoviral immunity. The vaccinated and challenged macaques had no signs of disease, and the invading pathogen rapidly cleared with no histopathological lesions. This is the first report showing the efficacy of an adenovirus-vectored trivalent vaccine against pneumonic plague in mouse and nonhuman primate (NHP) models.

New Role for FDA-Approved Drugs in Combating Antibiotic-Resistant Bacteria.

Antibiotic resistance in medically relevant bacterial pathogens, coupled with a paucity of novel antimicrobial discoveries, represents a pressing global crisis. Traditional drug discovery is an inefficient and costly process; however, systematic screening of Food and Drug Administration (FDA)-approved therapeutics for other indications in humans offers a rapid alternative approach. In this study, we screened a library of 780 FDA-approved drugs to identify molecules that rendered RAW 264.7 murine macrophages resistant to cytotoxicity induced by the highly virulent Yersinia pestis CO92 strain. Of these compounds, we identified 94 not classified as antibiotics as being effective at preventing Y. pestis-induced cytotoxicity. A total of 17 prioritized drugs, based on efficacy in in vitro screens, were chosen for further evaluation in a murine model of pneumonic plague to delineate if in vitro efficacy could be translated in vivo Three drugs, doxapram (DXP), amoxapine (AXPN), and trifluoperazine (TFP), increased animal survivability despite not exhibiting any direct bacteriostatic or bactericidal effect on Y. pestis and having no modulating effect on crucial Y. pestis virulence factors. These findings suggested that DXP, AXPN, and TFP may modulate host cell pathways necessary for disease pathogenesis. Finally, to further assess the broad applicability of drugs identified from in vitro screens, the therapeutic potential of TFP, the most efficacious drug in vivo, was evaluated in murine models of Salmonella enterica serovar Typhimurium and Clostridium difficile infections. In both models, TFP treatment resulted in increased survivability of infected animals. Taken together, these results demonstrate the broad applicability and potential use of nonantibiotic FDA-approved drugs to combat respiratory and gastrointestinal bacterial pathogens.

Cross-talk among flesh-eating Aeromonas hydrophila strains in mixed infection leading to necrotizing fasciitis.

Necrotizing fasciitis (NF) caused by flesh-eating bacteria is associated with high case fatality. In an earlier study, we reported infection of an immunocompetent individual with multiple strains of Aeromonas hydrophila (NF1-NF4), the latter three constituted a clonal group whereas NF1 was phylogenetically distinct. To understand the complex interactions of these strains in NF pathophysiology, a mouse model was used, whereby either single or mixed A. hydrophila strains were injected intramuscularly. NF2, which harbors exotoxin A (exoA) gene, was highly virulent when injected alone, but its virulence was attenuated in the presence of NF1 (exoA-minus). NF1 alone, although not lethal to animals, became highly virulent when combined with NF2, its virulence augmented by cis-exoA expression when injected alone in mice. Based on metagenomics and microbiological analyses, it was found that, in mixed infection, NF1 selectively disseminated to mouse peripheral organs, whereas the other strains (NF2, NF3, and NF4) were confined to the injection site and eventually cleared. In vitro studies showed NF2 to be more effectively phagocytized and killed by macrophages than NF1. NF1 inhibited growth of NF2 on solid media, but ExoA of NF2 augmented virulence of NF1 and the presence of NF1 facilitated clearance of NF2 from animals either by enhanced priming of host immune system or direct killing via a contact-dependent mechanism.

Intramuscular Immunization of Mice with a Live-Attenuated Triple Mutant of Yersinia pestis CO92 Induces Robust Humoral and Cell-Mediated Immunity To Completely Protect Animals against Pneumonic Plague.

Earlier, we showed that the Δlpp ΔmsbB Δail triple mutant of Yersinia pestis CO92 with deleted genes encoding Braun lipoprotein (Lpp), an acyltransferase (MsbB), and the attachment invasion locus (Ail), respectively, was avirulent in a mouse model of pneumonic plague. In this study, we further evaluated the immunogenic potential of the Δlpp ΔmsbB Δail triple mutant and its derivative by different routes of vaccination. Mice were immunized via the subcutaneous (s.c.) or the intramuscular (i.m.) route with two doses (2 × 10(6) CFU/dose) of the above-mentioned triple mutant with 100% survivability of the animals. Upon subsequent pneumonic challenge with 70 to 92 50% lethal doses (LD(50)) of wild-type (WT) strain CO92, all of the mice survived when immunization occurred by the i.m. route. Since Ail has virulence and immunogenic potential, a mutated version of Ail devoid of its virulence properties was created, and the genetically modified ail replaced the native ail gene on the chromosome of the Δlpp ΔmsbB double mutant, creating a Δlpp ΔmsbB::ailL2 vaccine strain. This newly generated mutant was attenuated similarly to the Δlpp ΔmsbB Δail triple mutant when administered by the i.m. route and provided 100% protection to animals against subsequent pneumonic challenge. Not only were the two above-mentioned mutants cleared rapidly from the initial i.m. site of injection in animals with no histopathological lesions, the immunized mice did not exhibit any disease symptoms during immunization or after subsequent exposure to WT CO92. These two mutants triggered balanced Th1- and Th2-based antibody responses and cell-mediated immunity. A substantial increase in interleukin-17 (IL-17) from the T cells of vaccinated mice, a cytokine of the Th17 cells, further augmented their vaccine potential. Thus, the Δlpp ΔmsbB Δail and Δlpp ΔmsbB::ailL2 mutants represent excellent vaccine candidates for plague, with the latter mutant still retaining Ail immunogenicity but with a much diminished virulence potential.