Next-generation nanovaccine induces durable immunity and protects against SARS-CoV-2.

While first generation SARS-CoV-2 vaccines were effective in slowing the spread and severity of disease during the COVID-19 pandemic, there is a need for vaccines capable of inducing durable and broad immunity against emerging variants of concern. Nanoparticle-based vaccines (i.e., "nanovaccines") composed of polyanhydride nanoparticles and pentablock copolymer micelles have previously been shown to protect against respiratory pathogens, including influenza A virus, respiratory syncytial virus, and Yersinia pestis. In this work, a nanovaccine containing SARS-CoV-2 spike and nucleocapsid antigens was designed and optimized. The optimized nanovaccine induced long-lived systemic IgG antibody responses against wild-type SARS-CoV-2 virus. In addition, the nanovaccine induced antibody responses capable of neutralization and cross-reactivity to multiple SARS-CoV-2 variants (including B.1.1.529) and antigen-specific CD4+ and CD8+ T cell responses. Finally, the nanovaccine protected mice against a lethal SARS-CoV-2 challenge, setting the stage for advancing particle-based SARS-CoV-2 nanovaccines. STATEMENT OF SIGNIFICANCE: First-generation SARS-CoV-2 vaccines were effective in slowing the spread and limiting the severity of COVID-19. However, current vaccines target only one antigen of the virus (i.e., spike protein) and focus on the generation of neutralizing antibodies, which may be less effective against new, circulating strains. In this work, we demonstrated the ability of a novel nanovaccine platform, based on polyanhydride nanoparticles and pentablock copolymer micelles, to generate durable and broad immunity against SARS-CoV-2. These nanovaccines induced long-lasting (> 62 weeks) serum antibody responses which neutralized binding to ACE2 receptors and were cross-reactive to multiple SARS-CoV-2 variants. Additionally, mice immunized with the SARS-CoV-2 nanovaccine showed a significant increase of antigen-specific T cell responses in the draining lymph nodes and spleens. Together, these nanovaccine-induced immune responses contributed to the protection of mice against a lethal challenge of live SARS-CoV-2 virus, indicating that this nanovaccine platform is a promising next-generation SARS-CoV-2 vaccine.

How do deer respiratory epithelial cells weather the initial storm of SARS-CoV-2 WA1/2020 strain?

The potential infectivity of severe acute respiratory syndrome associated coronavirus-2 (SARS-CoV-2) in animals raises a public health and economic concern, particularly the high susceptibility of white-tailed deer (WTD) to SARS-CoV-2. The disparity in the disease outcome between humans and WTD is very intriguing, as the latter are often asymptomatic, subclinical carriers of SARS-CoV-2. To date, no studies have evaluated the innate immune factors responsible for the contrasting SARS-CoV-2-associated disease outcomes in these mammalian species. A comparative transcriptomic analysis in primary respiratory epithelial cells of human (HRECs) and WTD (Deer-RECs) infected with the SARS-CoV-2 WA1/2020 strain was assessed throughout 48 h post inoculation (hpi). Both HRECs and Deer-RECs were susceptible to virus infection, with significantly (P < 0.001) lower virus replication in Deer-RECs. The number of differentially expressed genes (DEG) gradually increased in Deer-RECs but decreased in HRECs throughout the infection. The ingenuity pathway analysis of DEGs further identified that genes commonly altered during SARS-CoV-2 infection mainly belong to cytokine and chemokine response pathways mediated via interleukin-17 (IL-17) and nuclear factor-κB (NF-κB) signaling pathways. Inhibition of the NF-κB signaling in the Deer-RECs pathway was predicted as early as 6 hpi. The findings from this study could explain the lack of clinical signs reported in WTD in response to SARS-CoV-2 infection as opposed to the severe clinical outcomes reported in humans.IMPORTANCEThis study demonstrated that human and white-tailed deer primary respiratory epithelial cells are susceptible to the SARS-CoV-2 WA1/2020 strain infection. However, the comparative transcriptomic analysis revealed that deer cells could limit viral replication without causing hypercytokinemia by downregulating IL-17 and NF-κB signaling pathways. Identifying differentially expressed genes in human and deer cells that modulate key innate immunity pathways during the early infection will lead to developing targeted therapies toward preventing or mitigating the "cytokine storm" often associated with severe cases of coronavirus disease 19 (COVID-19). Moreover, results from this study will aid in identifying novel prognostic biomarkers in predicting SARS-CoV-2 adaption and transmission in deer and associated cervids.

Rapid detection of antimicrobial resistance in methicillin-resistant Staphylococcus aureus using MALDI-TOF mass spectrometry.

Antimicrobial resistance is a growing problem in modern healthcare. Most antimicrobial susceptibility tests (AST) require long culture times which delay diagnosis and effective treatment. Our group has previously reported a proof-of-concept demonstration of a rapid AST in Escherichia coli using deuterium labeling and MALDI mass spectrometry. Culturing bacteria in D2O containing media incorporates deuterium in newly synthesized lipids, resulting in a mass shift that can be easily detected by mass spectrometry. The extent of new growth is measured by the average mass of synthesized lipids that can be correlated with resistance in the presence of antimicrobials. In this work, we adapt this procedure to methicillin-resistant Staphylococcus aureus using the Bruker MALDI-TOF Biotyper, a low-cost instrument commonly available in diagnostic laboratories. The susceptible strain showed a significant decrease in average mass in on-target microdroplet cultures after 3 hours of incubation with 10 µg/mL methicillin, while the resistant strain showed consistent labeling regardless of methicillin concentration. This assay allows us to confidently detect methicillin resistance in S. aureus after only 3 hours of culture time and minimal sample processing, reducing the turn-around-time significantly over conventional assays. The success of this work suggests its potential as a rapid AST widely applicable in many clinical microbiology labs with minimal additional costs.

SARS-CoV-2 Is More Efficient than HCoV-NL63 in Infecting a Small Subpopulation of ACE2+ Human Respiratory Epithelial Cells.

Human coronavirus (HCoV)-NL63 is an important contributor to upper and lower respiratory tract infections, mainly in children, while severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, can cause lower respiratory tract infections, and more severe, respiratory and systemic disease, which leads to fatal consequences in many cases. Using microscopy, immunohistochemistry (IHC), virus-binding assay, reverse transcriptase qPCR (RT-qPCR) assay, and flow cytometry, we compared the characteristics of the susceptibility, replication dynamics, and morphogenesis of HCoV-NL63 and SARS-CoV-2 in monolayer cultures of primary human respiratory epithelial cells (HRECs). Less than 10% HRECs expressed ACE2, and SARS-CoV-2 seemed much more efficient than HCoV-NL63 at infecting the very small proportion of HRECs expressing the ACE2 receptors. Furthermore, SARS-CoV-2 replicated more efficiently than HCoV-NL63 in HREC, which correlates with the cumulative evidence of the differences in their transmissibility.

Data analytics-guided rational design of antimicrobial nanomedicines against opportunistic, resistant pathogens.

Nanoparticle carriers can improve antibiotic efficacy by altering drug biodistribution. However, traditional screening is impracticable due to a massive dataspace. A hybrid informatics approach was developed to identify polymer, antibiotic, and particle determinants of antimicrobial nanomedicine activity against Burkholderia cepacia, and to model nanomedicine performance. Polymer glass transition temperature, drug octanol-water partition coefficient, strongest acid dissociation constant, physiological charge, particle diameter, count and mass mean polydispersity index, zeta potential, fraction drug released at 2 h, and fraction release slope at 2 h were highly correlated with antimicrobial performance. Graph analysis provided dimensionality reduction while preserving nonlinear descriptor-property relationships, enabling accurate modeling of nanomedicine performance. The model successfully predicted particle performance in holdout validation, with moderate accuracy at rank-ordering. This data analytics-guided approach provides an important step toward the development of a rational design framework for antimicrobial nanomedicines against resistant infections by selecting appropriate carriers and payloads for improved potency.

Effective antiviral coatings for deactivating SARS-CoV-2 virus on N95 respirator masks or filters.

The application of antiviral coatings to masks and respirators is a potential mitigating step toward reducing viral transmission during the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic. The use of appropriate masks, social distancing, and vaccines is the immediate solution for limiting the viral spread and protecting people from this virus. N95 respirator masks are effective in filtering the virus particles, but they cannot kill or deactivate the virus. We report a possible approach to deactivating SARS-CoV-2 by applying an antimicrobial coating (Goldshield 75) to masks and respirators, rendering them suitable for repeated use. Masks coated with Goldshield 75 demonstrated continuous inactivation of the Alpha and Beta variants of the SARS-CoV-2 over a 3-day period and no loss of inactivation when stored at temperatures at 50 °C.

Enhanced apoptosis as a possible mechanism to self-limit SARS-CoV-2 replication in porcine primary respiratory epithelial cells in contrast to human cells.

The ability of SARS-CoV to infect different species, including humans, dogs, cats, minks, ferrets, hamsters, tigers, and deer, pose a continuous threat to human and animal health. Pigs, though closely related to humans, seem to be less susceptible to SARS-CoV-2. Former in vivo studies failed to demonstrate clinical signs and transmission between pigs, while later attempts using a higher infectious dose reported viral shedding and seroconversion. This study investigated species-specific cell susceptibility, virus dose-dependent infectivity, and infection kinetics, using primary human (HRECs) and porcine (PRECs) respiratory epithelial cells. Despite higher ACE2 expression in HRECs compared to PRECs, SARS-CoV-2 infected, and replicated in both PRECs and HRECs in a dose-dependent manner. Cytopathic effect was particularly more evident in PRECs than HRECs, showing the hallmark morphological signs of apoptosis. Further analysis confirmed an early and enhanced apoptotic mechanism driven through caspase 3/7 activation, limiting SARS-CoV-2 propagation in PRECs compared to HRECs. Our findings shed light on a possible mechanism of resistance of pigs to SARS-CoV-2 infection, and it may hold therapeutic value for the treatment of COVID-19.

Single-dose combination nanovaccine induces both rapid and durable humoral immunity and toxin neutralizing antibody responses against Bacillus anthracis.

Bacillus anthracis, the causative agent of anthrax, continues to be a prominent biological warfare and bioterrorism threat. Vaccination is likely to remain the most effective and user-friendly public health measure to counter this threat in the foreseeable future. The commercially available AVA BioThrax vaccine has a number of shortcomings where improvement would lead to a more practical and effective vaccine for use in the case of an exposure event. Identification of more effective adjuvants and novel delivery platforms is necessary to improve not only the effectiveness of the anthrax vaccine, but also enhance its shelf stability and ease-of-use. Polyanhydride particles have proven to be an effective platform at adjuvanting the vaccine-associated adaptive immune response as well as enhancing stability of encapsulated antigens. Another class of adjuvants, the STING pathway-targeting cyclic dinucleotides, have proven to be uniquely effective at inducing a beneficial inflammatory response that leads to the rapid induction of high titer antibodies post-vaccination capable of providing protection against bacterial pathogens. In this work, we evaluate the individual contributions of cyclic di-GMP (CDG), polyanhydride nanoparticles, and a combination thereof towards inducing neutralizing antibody (nAb) against the secreted protective antigen (PA) from B. anthracis. Our results show that the combination nanovaccine elicited rapid, high titer, and neutralizing IgG anti-PA antibody following single dose immunization that persisted for at least 108 DPI.

Induction of Potent and Durable Neutralizing Antibodies Against SARS-CoV-2 Using a Receptor Binding Domain-Based Immunogen.

A novel betacoronavirus (SARS-CoV-2) that causes severe pneumonia emerged through zoonosis in late 2019. The disease, referred to as COVID-19, has an alarming mortality rate and it is having a devastating effect on the global economy and public health systems. A safe, effective vaccine is urgently needed to halt this pandemic. In this study, immunogenicity of the receptor binding domain (RBD) of spike (S) glycoprotein was examined in mice. Animals were immunized with recombinant RBD antigen intraperitoneally using three different adjuvants (Zn-chitosan, Alhydrogel, and Adju-Phos), and antibody responses were followed for over 5 months. Results showed that potent neutralizing antibodies (nAbs) can be induced with 70% neutralization titer (NT70) of ~14,580 against live, infectious viruses. Although antigen-binding antibody titers decreased gradually over time, sufficiently protective levels of nAbs persisted (NT80 >2,430) over the 5-month observation period. Results also showed that adjuvants have profound effects on kinetics of nAb induction, total antibody titers, antibody avidity, antibody longevity, and B-cell epitopes targeted by the immune system. In conclusion, a recombinant subunit protein immunogen based on the RBD is a highly promising vaccine candidate. Continued evaluation of RBD immunogenicity using different adjuvants and vaccine regimens could further improve vaccine efficacy.

Single-dose combination nanovaccine induces both rapid and long-lived protection against pneumonic plague.

Yersinia pestis, the causative agent of pneumonic plague, induces a highly lethal infection if left untreated. Currently, there is no FDA-approved vaccine against this pathogen; however, USAMRIID has developed a recombinant fusion protein, F1-V, that has been shown to induce protection against pneumonic plague. Many F1-V-based vaccine formulations require prime-boost immunization to achieve protective immunity, and there are limited reports of rapid induction of protective immunity (≤ 14 days post-immunization (DPI)). The STimulator of INterferon Genes agonists cyclic dinucleotides (CDNs) have been shown to be promising vaccine adjuvants. Polyanhydride nanoparticle-based vaccines (i.e., nanovaccines) have also shown to enhance immune responses due to their dual functionality as adjuvants and delivery vehicles. In this work, a combination nanovaccine was designed that comprised F1-V-loaded nanoparticles combined with the CDN, dithio-RP,RP-cyclic di-guanosine monophosphate, to induce rapid and long-lived protective immunity against pneumonic plague. All mice immunized with a single dose combination nanovaccine were protected from Y. pestis lethal challenge within 14 DPI and demonstrated enhanced protection over F1-V adjuvanted with CDNs alone at challenge doses ≥7000 CFU Y. pestis CO92. In addition, 75% of mice receiving the single dose of the combination nanovaccine were protected from challenge at 182 DPI, while maintaining high levels of antigen-specific serum IgG. ELISPOT analysis of vaccinated animals at 218 DPI revealed F1-V-specific long-lived plasma cells in bone marrow in mice vaccinated with CDN adjuvanted F1-V or the combination nanovaccine. Microarray analysis of serum from these vaccinated mice revealed the presence of serum antibody that bound to a broad range of F1 and V linear epitopes. These results demonstrate that combining the adjuvanticity of CDNs with a nanovaccine delivery system enables induction of both rapid and long-lived protective immunity against Y. pestis. STATEMENT OF SIGNIFICANCE: • Yersinia pestis, the causative agent of pneumonic plague, induces a highly lethal infection if left untreated. Currently, there is no FDA-approved vaccine against this biodefense pathogen. • We designed a combination nanovaccine comprising of F1-V antigen-loaded polyanhydride nanoparticles and a cyclic dinucleotide adjuvant to induce both rapid and long-lived protective immunity against pneumonic plague. • Animals immunized with the combination nanovaccine maintained high levels of antigen-specific serum IgG and long-lived plasma cells in bone marrow and the serum antibody showed a high affinity for a broad range of F1 and V linear epitopes. • The combination nanovaccine is a promising next-generation vaccine platform against weaponized Y. pestis based on its ability to induce both rapid and long-lived protective immunity.

Data Analytics Approach for Rational Design of Nanomedicines with Programmable Drug Release.

Drug delivery vehicles can improve the functional efficacy of existing antimicrobial therapies by improving biodistribution and targeting. A critical property of such nanomedicine formulations is their ability to control the release kinetics of their payloads. The combination of (and interactions among) polymer, drug, and nanoparticle properties gives rise to nonlinear behavioral relationships and large data space. These factors complicate both first-principles modeling and screening of nanomedicine formulations. Predictive analytics may offer a more efficient approach toward the rational design of nanomedicines by identifying key descriptors and correlating them to nanoparticle release behavior. In this work, antibiotic release kinetics data were generated from polyanhydride nanoparticle formulations with varying copolymer compositions, encapsulated drug type, and drug loading. Four antibiotics, doxycycline, rifampicin, chloramphenicol, and pyrazinamide, were used. Linear manifold learning methods were used to relate drug release properties with polymer, drug, and nanoparticle properties, and key descriptors were identified that are highly correlated with release properties. However, these linear methods could not predict release behavior. Nonlinear multivariate modeling based on graph theory was then used to deconvolute the governing relationships between these properties, and predictive models were generated to rapidly screen lead nanomedicine formulations with desirable release properties with minimal nanoparticle characterization. Release kinetics predictions of two drugs containing atoms not included in the model showed good agreement with experimental results, validating the model and indicating its potential to virtually explore new polymer and drug pairs not included in the training data set. The models were shown to be robust after the inclusion of these new formulations, in that the new inclusions did not significantly change model regression. This approach provides the first step toward the development of a framework that can be used to rationally design nanomedicine formulations by selecting the appropriate carrier for a drug payload to program desirable release kinetics.

Antiparasitic Activity of Auranofin against Pathogenic Naegleria fowleri.

We report that the gold containing antirheumatoid drug auranofin is amoebicidal against human pathogenic Naegleria fowleri. Treatment of N. fowleri cultures at biologically relevant concentrations of 0.75-3.0 µg/ml auranofin reduced amoeba counts, metabolic activity, and increased cell permeability. These results suggest that the addition of auranofin may benefit the treatment of N. fowleri-infected patients afflicted by the rapidly fatal disease primary amoebic meningoencephalitis.

Nanotherapeutic provides dose sparing and improved antimicrobial activity against Brucella melitensis infections.

New therapies are needed to treat chronic bacterial diseases and intracellular pathogens, in particular, are very difficult to manage. The use of nanotherapeutics represents an approach to exploit size and charge of biological membranes to overcome barriers for treatment of intracellular pathogens including Brucella melitensis. In this work, polyanhydride nanoparticles comprised of copolymers of sebacic acid, 1,6-bis(p-carboxyphenoxy)hexane, and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane were synthesized to encapsulate antimicrobial compounds doxycycline and rifampicin. The nanoparticles demonstrated sustained release of rifampicin for a week with the antimicrobial activity peaking at 72 h and lasting up to a week. Treatment of B. melitensis infected macrophages with rifampicin-containing nanoparticles rapidly eliminated viable intracellular bacteria following 48 h of treatment and pretreatment with the nano-formulations prevented intracellular infection in contrast to soluble drug controls. Treatment of infected BALB/c mice with a nanoparticle cocktail containing doxycycline and rifampicin for five days decreased bacterial burden by three log10 in the liver. Extended release of antibiotics was demonstrated in vivo by treating B. melitensis infected mice with the standard therapy of daily 0.5 mg doxycycline dose or single 0.5 mg doxycycline-encapsulated nanoparticles delivered once a week. After 3 weeks, bacterial counts in spleen and liver were statistically equal between animals treated with the weekly nano-formulation and daily soluble drug, representing a seven-fold dose sparing. Altogether, these results demonstrated that the use of nanotherapeutics was successful at increasing antimicrobial efficacy and improving in vivo activity through a combination of intracellular delivery, dose sparing, and extended release in treating chronic bacterial infections. This platform technology can also provide benefits for drug delivery against other chronic intracellular bacterial pathogens, including Mycobacterium and Burkholderia species, including treatments against antibiotic-resistant infections.

Functionalization promotes pathogen-mimicking characteristics of polyanhydride nanoparticle adjuvants.

Rational design of adjuvants and delivery systems will promote development of next-generation vaccines to control emerging and re-emerging diseases. To accomplish this, understanding the immune-enhancing properties of new adjuvants relative to those induced by natural infections can help with the development of pathogen-mimicking materials that will effectively initiate innate immune signaling cascades. In this work, the surfaces of polyanhydride nanoparticles composed of sebacic acid (SA) and 1,6-bis(p-carboxyphenoxy) hexane were decorated with an ethylene diamine spacer partially modified with either a glycolic acid linker or an α-1,2-linked di-mannopyranoside (di-mannose) to confer "pathogen-like" properties and enhance adjuvanticity. Co-incubation of linker-modified nanoparticles with dendritic cells (DCs) elicited significant increases in surface expression of MHC I, MHC II, CD86, and CD40, and enhanced secretion of IL-6, IL-12p40, and TNF-α. An 800% increase in uptake of ethylene-diamine-spaced, linker and di-mannose functionalized polyanhydride nanoparticles was also observed. Together, our data showed that linker-functionalized polyanhydride nanoparticles demonstrate similar patterns of uptake, intracellular trafficking, particle persistence, and innate activation as did DCs exposed to Yersinia pestis or Escherichia coli. These results set the stage for rational selection of adjuvant chemistries to induce pathogen-mimicking immune responses. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2762-2771, 2017.

Cellular Internalization Mechanisms of Polyanhydride Particles: Implications for Rational Design of Drug Delivery Vehicles.

Polyanhydride nanoparticles have emerged as a versatile delivery platform, due to their ability to encapsulate diverse drugs, immunogens, antibodies, and proteins. However, mechanistic studies on the effects of particle chemistry interactions with immune cells have yet to be described. Understanding the mechanism by which these particles are internalized by immune cells will enable rational selection of delivery vehicles for specific applications. In the present study, the internalization, mechanism(s) of uptake by monocytes, and intracellular fate of polyanhydride nanoparticles were evaluated using copolymers based on 1,6-bis(p-carboxyphenoxy)hexane (CPH), sebacic acid (SA), and 1,8-bis(p-carboxyphenoxy)3,6-dioxaoctane (CPTEG). The results showed that 20:80 CPH:SA and 20:80 CPTEG:CPH nanoparticles were internalized to a greater extent by monocytes as compared to the 50:50 CPH:SA and 50:50 CPTEH:CPH nanoparticles. Further, cytochalasin-D treatment of cells inhibited uptake of all the particles, regardless of chemistry, indicating that actinmediated uptake is the primary mechanism of cellular entry for these particles. The insights gained from these studies were used to identify lead nanoparticle formulations to enhance treatment of intracellular bacterial infections. The use of doxycycline-loaded nanoparticles exhibited enhanced therapeutic efficacy compared to soluble drug in treating monocyte monolayers infected with the virulent intracellular pathogen Brucella abortus. Altogether, these studies demonstrate how rational design and selection of nanoscale delivery platforms can be used for a wide spectrum of biomedical applications.

Polyanhydride Nanoparticle Delivery Platform Dramatically Enhances Killing of Filarial Worms.

Filarial diseases represent a significant social and economic burden to over 120 million people worldwide and are caused by endoparasites that require the presence of symbiotic bacteria of the genus Wolbachia for fertility and viability of the host parasite. Targeting Wolbachia for elimination is a therapeutic approach that shows promise in the treatment of onchocerciasis and lymphatic filariasis. Here we demonstrate the use of a biodegradable polyanhydride nanoparticle-based platform for the co-delivery of the antibiotic doxycycline with the antiparasitic drug, ivermectin, to reduce microfilarial burden and rapidly kill adult worms. When doxycycline and ivermectin were co-delivered within polyanhydride nanoparticles, effective killing of adult female Brugia malayi filarial worms was achieved with approximately 4,000-fold reduction in the amount of drug used. Additionally the time to death of the macrofilaria was also significantly reduced (five-fold) when the anti-filarial drug cocktail was delivered within polyanhydride nanoparticles. We hypothesize that the mechanism behind this dramatically enhanced killing of the macrofilaria is the ability of the polyanhydride nanoparticles to behave as a Trojan horse and penetrate the cuticle, bypassing excretory pumps of B. malayi, and effectively deliver drug directly to both the worm and Wolbachia at high enough microenvironmental concentrations to cause death. These provocative findings may have significant consequences for the reduction in the amount of drug and the length of treatment required for filarial infections in terms of patient compliance and reduced cost of treatment.

Nano-enabled delivery of diverse payloads across complex biological barriers.

Complex biological barriers are major obstacles for preventing and treating disease. Nanocarriers are designed to overcome such obstacles by enhancing drug delivery through physiochemical barriers and improving therapeutic indices. This review critically examines both biological barriers and nanocarrier payloads for a variety of drug delivery applications. A spectrum of nanocarriers is discussed that have been successfully developed for improving tissue penetration for preventing or treating a range of infectious, inflammatory, and degenerative diseases.

An in vitro model of antibody-enhanced killing of the intracellular parasite Leishmania amazonensis.

Footpad infection of C3HeB/FeJ mice with Leishmania amazonensis leads to chronic lesions accompanied by large parasite loads. Co-infecting these animals with L. major leads to induction of an effective Th1 immune response that can resolve these lesions. This cross-protection can be recapitulated in vitro by using immune cells from L. major-infected animals to effectively activate L. amazonensis-infected macrophages to kill the parasite. We have shown previously that the B cell population and their IgG2a antibodies are required for effective cross-protection. Here we demonstrate that, in contrast to L. major, killing L. amazonensis parasites is dependent upon FcRγ common-chain and NADPH oxidase-generated superoxide from infected macrophages. Superoxide production coincided with killing of L. amazonensis at five days post-activation, suggesting that opsonization of the parasites was not a likely mechanism of the antibody response. Therefore we tested the hypothesis that non-specific immune complexes could provide a mechanism of FcRγ common-chain/NADPH oxidase dependent parasite killing. Macrophage activation in response to soluble IgG2a immune complexes, IFN-γ and parasite antigen was effective in significantly reducing the percentage of macrophages infected with L. amazonensis. These results define a host protection mechanism effective during Leishmania infection and demonstrate for the first time a novel means by which IgG antibodies can enhance killing of an intracellular pathogen.

Functionalization of polyanhydride microparticles with di-mannose influences uptake by and intracellular fate within dendritic cells.

Innovative vaccine delivery platforms can facilitate the development of effective single-dose treatment regimens to control emerging and re-emerging infectious diseases. Polyanhydride microparticles are promising vaccine delivery vehicles due to their ability to stably maintain antigens, provide tailored release kinetics and function as adjuvants. A major obstacle for the use of microparticle-based vaccines, however, is their limited uptake by dendritic cells (DCs). In this study, we functionalized the microparticle surface with di-mannose in order to target C-type lectin receptors (CLRs) on DCs. Polyanhydride particles based on sebacic acid (SA), 1,6-bis(p-carboxyphenoxy)hexane (CPH) and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) were evaluated. Co-incubation of di-mannose-functionalized microparticles up-regulated the expression of CLRs on DCs. More importantly, di-mannose functionalization increased the uptake, as measured by the percentage of cells internalizing particles. The uptake of CPH:SA microparticles increased ∼20-fold, from 0.82% (non-functionalized) to 20.2%, and internalization of CPTEG:CPH microparticles increased ∼7-fold from 1.35% (non-functionalized) to 9.3% upon di-mannose functionalization. Both di-mannose-functionalized and non-functionalized particles trafficked to lysosomes. Together, these studies demonstrate that employing rational vaccine design principles, such as the targeting of CLRs on antigen-presenting cells, can enhance delivery of encapsulated antigens and potentially induce a more robust adaptive immune response.

Host interferon-γ inducible protein contributes to Brucella survival.

Brucella spp. are highly adapted intracellular pathogens of mammals that cause chronic infections while surving and replicating in host monocytes and macrophages. Although monocytes are normally susceptible to infection, pretreatment with pro-inflammatory cytokine interferon-γ (IFN-γ) activates cellular defense mechanisms that increase intracellular killing of Brucella and prevents bacterial replication. We examined the contribution of the IFN-γ inducible GTPase, LRG-47, to B. abortus 2308 infection in in vitro and in vivo murine models. Infecting non-activated macrophages from LRG-47(-/-) mice revealed that loss of this host protein negatively effected the intracellular survival and replication of IgG opsonized B. abortus. In contrast, survival and replication of non-opsonized B. abortus was the same in both C57/B6 and LRG-47(-/-) peritoneal macrophages. Following IFN-γ activation of LRG-47(-/-) monocytes, IgG opsonized B. abortus survived better than non-opsonized bacteria. The differential fate of opsonized and non-opsonized B. abortus was only observed in macrophages collected from LRG-47(-/-) mice. Given the specific nature of the relationship between this host protein and the mechanism of Brucella internalization, LRG-47(-/-) mice were infected with B. abortus to assess whether the loss of the lrg47 protein would affect the ability of the bacteria to colonize or persist within the host. B. abortus were able to establish and maintain similar numbers of bacteria in both C57/B6 mice and LRG-47(-/-) through 3 weeks post intraperitoneal infection. By 9 weeks p.i. fewer B. abortus were recovered from LRG-47(-/-) mice than controls, suggesting that the host protein has a positive role in maintaining long term persistence of the bacteria within the host. These observations demonstrating a positive role for a host IFN-γ induced protein defense protein has yet to be reported. These results provide interesting insight into the complex interaction between Brucella and their host.