Insights into mechanisms of antibody-mediated immunity from studies with Cryptococcus neoformans

At first glance Cryptococcus neoformans appears an unlikely microbe to provide a new understanding of mechanisms of antibody-mediated immunity (AMI), because it is a facultative intracellular fungal pathogen for which the role of naturally acquired AMI in host defense is uncertain. However, numerous studies have now established that certain antibodies (Abs) against C. neoformans are protective in certain hosts. Studies with Abs to C. neoformans have provided new insights into AMI and generated new precedents with implications for other pathogens. The following concepts have emerged: 1) susceptibility to C. neoformans may be related to qualitative and quantitative aspects of the Ab response; 2) protective monoclonal Abs can be generated against pathogens even when the role of humoral immunity is uncertain; 3) Abs to C. neoformans mediate protection by immunomodulatory effects, thereby linking Ab efficacy to the overall host immune response; 4) Ab efficacy is critically dependent on fine specificity, which in turn is affected by immunoglobulin variable region usage, somatic mutation and constant region usage; 5) the efficacy of passive Ab therapy is a function of Ab dose and infecting innoculum, with lack of efficacy at the extremes of Ab concentration; 6) Ab-mediated toxicity resulting from antigen-Ab complex-induced release of platelet activating factor is isotype dependent. Observations with C. neoformans have stimulated a reappraisal of the role of humoral immunity for other pathogens and highlighted the limitations in current methods of assessing the role of Ab in host defense.     

Protective Immunity against Trichinella spiralis Infection Induced by a Multi-Epitope Vaccine in a Murine Model

Trichinellosis is one of the most important food-borne parasitic zoonoses throughout the world. Because infected pigs are the major source of human infections, and China is becoming the largest international producer of pork, the development of a transmission-blocking vaccine to prevent swine from being infected is urgently needed for trichinellosis control in China. Our previous studies have demonstrated that specific Trichinella spiralis paramyosin (Ts-Pmy) and Ts-87 antigen could provide protective immunity against T. spiralis infection in immunized mice. Certain protective epitopes of Ts-Pmy and Ts-87 antigen have been identified. To identify more Ts-Pmy protective epitopes, a new monoclonal antibody, termed 8F12, was produced against the N-terminus of Ts-Pmy. This antibody elicited significant protective immunity in mice against T. spiralis infection by passive transfer and was subsequently used to screen a random phage display peptide library to identify recognized epitopes. Seven distinct positive phage clones were identified and their displayed peptides were sequenced. Synthesized epitope peptides conjugated to keyhole limpet hemocyanin were used to immunize mice, four of which exhibited larval reduction (from 18.7% to 26.3%, respectively) in vaccinated mice in comparison to the KLH control. To increase more effective protection, the epitope 8F7 that was found to induce the highest protection in this study was combined with two other previously identified epitopes (YX1 from Ts-Pmy and M7 from Ts-87) to formulate a multi-epitope vaccine. Mice immunized with this multi-epitope vaccine experienced a 35.0% reduction in muscle larvae burden after being challenged with T. spiralis larvae. This protection is significantly higher than that induced by individual-epitope peptides and is associated with high levels of subclasses IgG and IgG1. These results showed that a multi-epitope vaccine induced better protective immunity than an individual epitope and provided a feasible approach for developing a safer and more effective vaccine against trichinellosis.     

IL-4 Receptor-Alpha-Dependent Control of Cryptococcus neoformans in the Early Phase of Pulmonary Infection

Cryptococcus neoformans is an opportunistic fungal pathogen that causes lung inflammation and meningoencephalitis in immunocompromised people. Previously we showed that mice succumb to intranasal infection by induction of pulmonary interleukin (IL)-4Rα–dependent type 2 immune responses, whereas IL-12-dependent type 1 responses confer resistance. In the experiments presented here, IL-4Rα^−/− mice unexpectedly show decreased fungal control early upon infection with C. neoformans, whereas wild-type mice are able to control fungal growth accompanied by enhanced macrophage and dendritic cell recruitment to the site of infection. Lower pulmonary recruitment of macrophages and dendritic cells in IL-4Rα^−/− mice is associated with reduced pulmonary expression of CCL2 and CCL20 chemokines. Moreover, IFN-γ and nitric oxide production are diminished in IL-4Rα^−/− mice compared to wild-type mice. To directly study the potential mechanism(s) responsible for reduced production of IFN-γ, conventional dendritic cells were stimulated with C. neoformans in the presence of IL-4 which results in increased IL-12 production and reduced IL-10 production. Together, a beneficial role of early IL-4Rα signaling is demonstrated in pulmonary cryptococcosis, which contrasts with the well-known IL-4Rα-mediated detrimental effects in the late phase.     

Mouse genetic background is a major determinant of isotype-related differences for antibody-mediated protective efficacy against Cryptococcus neoformans

The protective efficacy of mAbs to Cryptococcus neoformans glucuronoxylomannan depends on Ab isotype. Previous studies in A/JCr and C57BL/6J mice showed relative protective efficacy of IgG1, IgG2a > IgG3. However, we now report that in C57BL/6J x 129/Sv mice, IgG3 is protective while IgG1 is not protective, with neither isotype being protective in 129/Sv mice. IgG1, IgG2a, and IgG3 had different effects on IFN-gamma expression in infected C57BL/6J x 129/Sv mice. IgG1-treated C57BL/6J x 129/Sv mice had significantly more pulmonary eosinophilia than IgG2a- and IgG3-treated C57BL/6J x 129/Sv mice. C. neoformans infection and Ab administration had different effects on FcgammaRI, FcgammaRII, and FcgammaRIII expression in C57BL/6J, 129/Sv, and C57BL/6J x 129/Sv mice. Our results indicate that the relative efficacy of Ab isotype function against C. neoformans is a function of the genetic background of the host and that IgG3-mediated protection in C57BL/6J x 129/Sv mice was associated with lower levels of IFN-gamma and fewer pulmonary eosinophils. The dependence of isotype efficacy on host genetics underscores a previously unsuspected complex relationship between the cellular and humoral arms of the adaptive immune response.     

Pathogenesis of Pulmonary Cryptococcus gattii Infection: A Rat Model

A model of pulmonary cryptococcosis in immunocompetent rats was developed to better understand the virulence of Cryptococcus gattii. Six isolates were studied, representing four molecular genotypes (VGI-MATα, VGIIa-MATα, VGIIa-MAT a, VGIIb-MATα), obtained from Australia, Vancouver (Canada) and Colombia. These originated from human patients, a cat and the environment and were administered intratracheally (i.t.) or transthoracically into Fischer 344 or Wistar-Furth rats in doses varying from 10⁴ to 10⁷ colony-forming units (CFU) in 0.1 ml of saline. With the exception of animals given the VGIIa-MAT a isolate, rats consistently became ill or died of progressive cryptococcal pneumonia following i.t. doses exceeding 10⁷ CFU. Affected lungs increased in weight up to tenfold and contained numerous circumscribed, gelatinous lesions. These became larger and more extensive, progressing from limited hilar and/or tracheal lesions, to virtually confluent gelatinous masses. Disease was localized to the lungs for at least 3–4 weeks, with dissemination to the brain occurring in some animals after day 29. The dose–response relationship was steep for two VGI isolates studied (human WM179, environmental WM276); doses up to 10⁶ CFU i.t. did not produce lesions, while 10⁷ or more yeast cells produced progressive pneumonia. Intratracheal inoculation of rats with C. gattii provides an excellent model of human pulmonary cryptococcosis in healthy hosts, mimicking natural infections. Disease produced by C. gattii in rats is distinct from that caused by C. neoformans in that infections are progressive and ultimately fatal.     

Mechanisms of Immunity in Post-Exposure Vaccination against Ebola Virus Infection

Ebolaviruses can cause severe hemorrhagic fever that is characterized by rapid viral replication, coagulopathy, inflammation, and high lethality rates. Although there is no clinically proven vaccine or treatment for Ebola virus infection, a virus-like particle (VLP) vaccine is effective in mice, guinea pigs, and non-human primates when given pre-infection. In this work, we report that VLPs protect Ebola virus-infected mice when given 24 hours post-infection. Analysis of cytokine expression in serum revealed a decrease in pro-inflammatory cytokine and chemokine levels in mice given VLPs post-exposure compared to infected, untreated mice. Using knockout mice, we show that VLP-mediated post-exposure protection requires perforin, B cells, macrophages, conventional dendritic cells (cDCs), and either CD4+ or CD8+ T cells. Protection was Ebola virus-specific, as marburgvirus VLPs did not protect Ebola virus-infected mice. Increased antibody production in VLP-treated mice correlated with protection, and macrophages were required for this increased production. However, NK cells, IFN-gamma, and TNF-alpha were not required for post-exposure-mediated protection. These data suggest that a non-replicating Ebola virus vaccine can provide post-exposure protection and that the mechanisms of immune protection in this setting require both increased antibody production and generation of cytotoxic T cells.     

The Role of Cryptococcus in the Immune System of Pulmonary Cryptococcosis Patients

Objectives

To investigate the role of Cryptococcus in the immune system of immunocompetent patients with pulmonary cryptococcosis (PC) by analysing the dynamic changes of patients’ immune status before and after antifungal therapy.

Methods

The level of the serum interferon-γ (IFN-γ) and interleukin (IL)-2, -4, -10 and -12 was measured before and after 6-months of treatment. Peripheral blood samples were obtained from 30 immunocompetent PC patients and 30 age- and gender-matched healthy controls. Peripheral blood mononuclear cells (PBMCs) were isolated and incubated with recombinant human IL-12 (rhIL-12) for 48 h. Then the concentrations of IFN-γ and IL-4 in the supernatant were analysed.

Results

Baseline serum IFN-γ level was significantly lower in the PC patients as compared with the control group (P < 0.001). The serum IL-2 and IFN-γ of PC patients were significantly increased after appropriate treatments (P < 0.05 and P < 0.001 when compared to their baseline levels). The productions of IFN-γ in the culture supernatant of PBMCs showed no significant difference between the control and PC patients both before and after antifungal treatments. RhIL-12 is a potent stimulus for IFN-γ production. Culture PBMCs collected from PC patients before treatments had a smaller increase of IFN-γ production in the present of rhIL-12 than the control (P < 0.01); PBMCs from PC patients completing 6-months of treatment showed a comparable increase of IFN-γ production by rhIL-12 stimulation to the control group.

Conclusions

In apparently immunocompetent patients with PC, a normalization of serum IFN-γ was achieved after recovery from infection. This suggests that Cryptococcus infection per se can suppress the immune system and its elimination contributes to the reestablishment of an immune equilibrium.     

Insights into Persistence Mechanisms of a Zoonotic Virus in Bat Colonies Using a Multispecies Metapopulation Model

Rabies is a worldwide zoonosis resulting from Lyssavirus infection. In Europe, Eptesicus serotinus is the most frequently reported bat species infected with Lyssavirus, and thus considered to be the reservoir of European bat Lyssavirus type 1 (EBLV-1). To date, the role of other bat species in EBLV-1 epidemiology and persistence remains unknown. Here, we built an EBLV-1−transmission model based on local observations of a three-cave and four-bat species (Myotis capaccinii, Myotis myotis, Miniopterus schreibersii, Rhinolophus ferrumequinum) system in the Balearic Islands, for which a 1995–2011 serological dataset indicated the continuous presence of EBLV-1. Eptesicus serotinus was never observed in the system during the 16-year follow-up and therefore was not included in the model. We used the model to explore virus persistence mechanisms and to assess the importance of each bat species in the transmission dynamics. We found that EBLV-1 could not be sustained if transmission between M. schreibersii and other bat species was eliminated, suggesting that this species serves as a regional reservoir. Global sensitivity analysis using Sobol''s method revealed that following the rate of autumn−winter infectious contacts, M. schreibersii''s incubation- and immune-period durations, but not the infectious period length, were the most relevant factors driving virus persistence.     

Immunization with Lipopolysaccharide-Deficient Whole Cells Provides Protective Immunity in an Experimental Mouse Model of Acinetobacter baumannii Infection

The increasing clinical importance of infections caused by multidrug resistant Acinetobacter baumannii warrants the development of novel approaches for prevention and treatment. In this context, vaccination of certain patient populations may contribute to reducing the morbidity and mortality caused by this pathogen. Vaccines against Gram-negative bacteria based on inactivated bacterial cells are highly immunogenic and have been shown to produce protective immunity against a number of bacterial species. However, the high endotoxin levels present in these vaccines due to the presence of lipopolysaccharide complicates their use in human vaccination. In the present study, we used a laboratory-derived strain of A. baumannii that completely lacks lipopolysaccharide due to a mutation in the lpxD gene (IB010), one of the genes involved in the first steps of lipopolysaccharide biosynthesis, for vaccination. We demonstrate that IB010 has greatly reduced endotoxin content (<1.0 endotoxin unit/10⁶ cells) compared to wild type cells. Immunization with formalin inactivated IB010 produced a robust antibody response consisting of both IgG1 and IgG2c subtypes. Mice immunized with IB010 had significantly lower post-infection tissue bacterial loads and significantly lower serum levels of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6 compared to control mice in a mouse model of disseminated A. baumannii infection. Importantly, immunized mice were protected from infection with the ATCC 19606 strain and an A. baumannii clinical isolate. These data suggest that immunization with inactivated A. baumannii whole cells deficient in lipopolysaccharide could serve as the basis for a vaccine for the prevention of infection caused by A. baumannii.     

CD47 Promotes Protective Innate and Adaptive Immunity in a Mouse Model of Disseminated Candidiasis

CD47 is a widely expressed receptor that regulates immunity by engaging its counter-receptor SIRPα on phagocytes and its secreted ligand thrombospondin-1. Mice lacking CD47 can exhibit enhanced or impaired host responses to bacterial pathogens, but its role in fungal immunity has not been examined. cd47^-/- mice on a C57BL/6 background showed significantly increased morbidity and mortality following Candida albicans infection when compared with wild-type mice. Despite normal fungal colonization at earlier times, cd47^-/- mice at four days post-infection had increased colonization of brain and kidneys accompanied by stronger inflammatory reactions. Neutrophil and macrophage numbers were significantly elevated in kidneys and neutrophils in the brains of infected cd47^-/- mice. However, no defect in phagocytic activity towards C. albicans was observed in cd47^-/- bone-marrow-derived macrophages, and neutrophil and macrophage killing of C. albicans was not impaired. CD47-deficiency did not alter the early humoral immune response to C. albicans. Th1, Th2, and Th17 population of CD4⁺ T cells were expanded in the spleen, and gene expression profiles of spleen and kidney showed stronger pro-inflammatory signaling in infected cd47^-/- mice. The chemoattractant chemokines MIP-2α and MIP-2β were highly expressed in infected spleens of cd47^-/- mice. G-CSF, GM-CSF, and the inflammasome component NLRP3 were more highly expressed in infected cd47^-/- kidneys than in infected wild-type controls. Circulating pro- (TNF-α, IL-6) and anti-inflammatory cytokines (IL-10) were significantly elevated, but IL-17 was decreased. These data indicate that CD47 plays protective roles against disseminated candidiasis and alters pro-inflammatory and immunosuppressive pathways known to regulate innate and T cell immunity.     

Effects of microplusin, a copper-chelating antimicrobial peptide, against Cryptococcus neoformans

Microplusin is an antimicrobial peptide isolated from the cattle tick Rhipicephalus (Boophilus) microplus. Its copper-chelating ability is putatively responsible for its bacteriostatic activity against Micrococcus luteus as microplusin inhibits respiration in this species, which is a copper-dependent process. Microplusin is also active against Cryptococcus neoformans (MIC(50) = 0.09 μM), the etiologic agent of cryptococcosis. Here, we show that microplusin is fungistatic to C. neoformans and this inhibitory effect is abrogated by copper supplementation. Notably, microplusin drastically altered the respiratory profile of C. neoformans. In addition, microplusin affects important virulence factors of this fungus. We observed that microplusin completely inhibited fungal melanization, and this effect correlates with the inhibition of the related enzyme laccase. Also, microplusin significantly inhibited the capsule size of C. neoformans. Our studies reveal, for the first time, a copper-chelating antimicrobial peptide that inhibits respiration and growth of C. neoformans and modifies two major virulence factors: melanization and formation of a polysaccharide capsule. These features suggest that microplusin, or other copper-chelation approaches, may be a promising therapeutic for cryptococcosis.     

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient’s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.     

Evaluation of the Protective Immunity of a Novel Subunit Fusion Vaccine in a Murine Model of Systemic MRSA Infection

Staphylococcus aureus is a common commensal organism in humans and a major cause of bacteremia and hospital acquired infection. Because of the spread of strains resistant to antibiotics, these infections are becoming more difficult to treat. Therefore, exploration of anti-staphylococcal vaccines is currently a high priority. Iron surface determinant B (IsdB) is an iron-regulated cell wall-anchored surface protein of S. aureus. Alpha-toxin (Hla) is a secreted cytolytic pore-forming toxin. Previous studies reported that immunization with IsdB or Hla protected animals against S. aureus infection. To develop a broadly protective vaccine, we constructed chimeric vaccines based on IsdB and Hla. Immunization with the chimeric bivalent vaccine induced strong antibody and T cell responses. When the protective efficacy of the chimeric bivalent vaccine was compared to that of individual proteins in a murine model of systemic S. aureus infection, the bivalent vaccine showed a stronger protective immune response than the individual proteins (IsdB or Hla). Based on the results presented here, the chimeric bivalent vaccine affords higher levels of protection against S. aureus and has potential as a more effective candidate vaccine.     

Profiling a killer, the development of Cryptococcus neoformans

The ability of fungi to transition between unicellular and multicellular growth has a profound impact on our health and the economy. Many important fungal pathogens of humans, animals, and plants are dimorphic, and the ability to switch between morphological states has been associated with their virulence. Cryptococcus neoformans is a human fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised and, in some cases, immunocompetent hosts. Cryptococcus neoformans grows vegetatively as a budding yeast and switches to hyphal growth during the sexual cycle, which is important in the study of cryptococcal pathogenicity because spores resulting from sexual development are infectious propagules and can colonize the lungs of a host. In addition, sexual reproduction contributes to the genotypic variability of Cryptococcus species, which may lead to increased fitness and virulence. Despite significant advances in our understanding of the mechanisms behind the development of C. neoformans, our knowledge is still incomplete. Recent studies have led to the emergence of many intriguing questions and hypotheses. In this review, we describe and discuss the most interesting aspects of C. neoformans development and address their impact on pathogenicity.     

In vitro activity of a liposomal nystatin formulation (Nyotran) against Cryptococcus neoformans

The in vitro antifungal activity of a new liposomal nystatin formulation (NISTL, Nyotran, Aronex Ltd., EE.UU.) was evaluated by a microdilution method with RPMI based on the M27A document of the National Committee for Clinical Laboratory Standards (NCCLS) against 22 isolates of Cryptococcus neoformans. This antifungal activity was compared with those of other seven antifungal agents, such as nystatin (NIST), amphotericin B deoxycholate, liposomal amphotericin B, amphotericin B lipid complex, amphotericin B colloidal dispersion, fluconazole, and itraconazole. NISTL was more active in vitrothan NIST, showing MIC values 2-3 fold smaller in 90% of the isolates. The results obtained suggest that this new formulation would be very helpful for the treatment of cryptococcosis.     

利用雌激素构建小鼠淋病奈瑟菌感染模型

目的探讨用雌激素构建淋病奈瑟菌感染小鼠模型的方法。方法淋病奈瑟菌WHO-A接种经皮下注射苯甲酸雌二醇和真皮下埋植尼尔雌醇片（雌三醇）预处理的雌性ICR小鼠,小鼠阴道拭子接种T-M选择培养基培养分离淋病奈瑟菌,取小鼠阴道分泌物涂片染色观察淋病奈瑟菌感染情况,比较两种雌激素对小鼠淋病奈瑟菌感染的差异。结果与对照组相比,苯甲酸雌二醇小鼠体内淋病奈瑟菌有短时间的定植,而尼尔雌醇组与对照组无差异。结论雌激素（尤其是雌二醇）有利于淋病奈瑟菌在小鼠阴道内的定植。     

Role of sphingomyelin synthase in controlling the antimicrobial activity of neutrophils against Cryptococcus neoformans

The key host cellular pathway(s) necessary to control the infection caused by inhalation of the environmental fungal pathogen Cryptococcus neoformans are still largely unknown. Here we have identified that the sphingolipid pathway in neutrophils is required for them to exert their killing activity on the fungus. In particular, using both pharmacological and genetic approaches, we show that inhibition of sphingomyelin synthase (SMS) activity profoundly impairs the killing ability of neutrophils by preventing the extracellular release of an antifungal factor(s). We next found that inhibition of protein kinase D (PKD), which controls vesicular sorting and secretion and is regulated by diacylglycerol (DAG) produced by SMS, totally blocks the extracellular killing activity of neutrophils against C. neoformans. The expression of SMS genes, SMS activity and the levels of the lipids regulated by SMS (namely sphingomyelin (SM) and DAG) are up-regulated during neutrophil differentiation. Finally, tissue imaging of lungs infected with C. neoformans using matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS), revealed that specific SM species are associated with neutrophil infiltration at the site of the infection. This study establishes a key role for SMS in the regulation of the killing activity of neutrophils against C. neoformans through a DAG-PKD dependent mechanism, and provides, for the first time, new insights into the protective role of host sphingolipids against a fungal infection.