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A molecule we termed Brugia malayi IL-5 receptor (IL-5R) binding protein (BmIL5Rbp; also known as Bm8757) was identified from B. malayi filarial worms and found to inhibit human interleukin-5 (IL-5) binding to its human receptor competitively. After the expression and purification of a recombinant BmIL5Rbp and generation of BmIL5Rbp-specific rabbit antibody, we localized the molecule on B. malayi worms through immunohistochemistry and immunoelectron microscopy. RNA interference (RNAi) was used to inhibit BmIL5Rbp mRNA and protein production. BmIL5Rbp was shown to localize to the cuticle of Brugia malayi and to be released in its excretory/secretory products. RNAi inhibited BmIL5Rbp mRNA production by 33%, reduced the surface protein expression by ~50%, and suppressed the release of BmIL5Rbp in the excretory/secretory products. RNAi has been used successfully to knock down the mRNA and protein expression of BmIL5Rbp in the early larval stages of B. malayi and provided a proof of principle for the local inhibition of the human IL-5R. These findings provide evidence that a parasite-encoded IL-5R antagonist may locally inhibit a vital host innate immune activation of IL-5 on eosinophils.

Millions of people worldwide are infected with filarial nematodes, responsible for lymphatic filariasis (LF) and other diseases causing chronic disablement. Elimination programs have resulted in a substantial reduction of the rate of infection in certain areas creating a need for improved diagnostic tools to establish robust population surveillance and avoid LF resurgence. Glycans from parasitic helminths are emerging as potential antigens for use in diagnostic assays. However, despite its crucial role in host- parasite interactions, filarial glycosylation is still largely, structurally, and functionally uncharacterized. Therefore, we investigated the glycan repertoire of the filarial nematode Brugia malayi. Glycosphingolipid and N-linked glycans were extracted from several life-stages using enzymatic release and characterized using a combination of MALDITOF-MS and glycan sequencing techniques. Next, glycans were purified by HPLC and printed onto microarrays to assess the host anti-glycan antibody response. Comprehensive glycomic analysis of B. malayi revealed the presence of several putative antigenic motifs such as phosphorylcholine and terminal glucuronic acid. Glycan microarray screening showed a recognition of most B. malayi glycans by immunoglobulins from rhesus macaques at different time points after infection, which permitted the characterization of the dynamics of anti-glycan immunoglobulin G and M during the establishment of brugian filariasis. A significant level of IgG binding to the parasite glycans was also detected in infected human plasma, while IgG binding to glycans decreased after anthelmintic treatment. Altogether, our work identifies B. malayi glycan antigens and reveals antibody responses from the host that could be exploited as potential markers for LF.

A molecule termed BmIL5Rbp (aka Bm8757) was identified from Brugia malayi filarial worms and found to competitively inhibit human IL-5 binding to its human receptor. After the expression and purification of a recombinant BmIL5Rbp and generation of BmIL5Rbp-specific rabbit antibody, we localized the molecule on B. malayi worms through immunohistochemistry and immunoelectron microscopy. RNA interference was used to inhibit BmIL5Rbp mRNA and protein production. BmIL5Rbp was shown to localize to the cuticle of Brugia malayi and to be released in their excretory/secretory products. RNAi inhibited BmIL5Rbp mRNA production by 33% and reduced the surface protein expression by ~50% and suppressed the release of BmIL5Rbp in the excretory/secretory products. RNAi has been used successfully to knock down the mRNA and protein expression of BmIL5Rbp in the early larval stages of B. malayi and provided a proof-of-principle for the local inhibition of the human IL5 receptor. These findings provide evidence that a parasite encoded IL5R antagonist could be utilized therapeutically.

Inflammatory bowel disease (IBD) remains a persistent health problem with a global burden surging over 6.8 million cases currently. Clinical pathology of IBD is complicated; however, hyperactive inflammatory and immune responses in the gut is shown to be one of the persistent causes of the disease. Human gut inflammasome, the activator of innate immune system is believed to be a primary underlying cause for the pathology and is largely associated with the progression of IBD. To manage IBD, there is a need to fully understand the role of inflammasome activation in IBD. Since inflammasome potentially play a significant role in IBD, systemic modulation of inflammasome may provide an effective therapeutic and clinical approach to control IBD symptoms. In this review, we have focused on this association between IBD and gut inflammasome, and recent advances in the research and therapeutic strategies for IBD. We have discussed inflammasomes and their components, outcomes from the experimental animals and human studies, inflammasome inhibitors, and developments in the inflammasome-targeted therapies for IBD.

Immunomodulatory molecules produced by helminth parasites are receiving much attention recently as novel therapeutic agents for inflammation and autoimmune diseases. In this study, we show that macrophage migration inhibitory factor (MIF) homologue from the filarial parasite, Wuchereria bancrofti (rWbaMIF-2), can suppress inflammation in a dextran sulphate sodium salt (DSS)-induced colitis model. The disease activity index (DAI) in DSS given mice showed loss of body weight and bloody diarrhoea. At autopsy, colon of these mice showed severe inflammation and reduced length. Administration of rWbaMIF-2 significantly reduced the DAI in DSS-induced colitis mice. rWbaMIF-2-treated mice had no blood in the stools, and their colon length was similar to the normal colon with minimal inflammation and histological changes. Pro-inflammatory cytokine genes (TNF-α, IL-6, IFN-γ, IL-1β, IL-17A and NOS2) were downregulated in the colon tissue and peritoneal macrophages of rWbaMIF-2-treated mice. However, there were significant increases in IL-10-producing Treg and B1 cells in the colon and peritoneal cavity of rWbaMIF-2-treated mice. These findings suggested that rWbaMIF-2 treatment significantly ameliorated the clinical symptoms, inflammation and colon pathology in DSS given mice. This immunomodulatory effect of rWbaMIF-2 appeared to be by promoting the infiltration of Treg cells into the colon.

Introduction: We recently showed in a rodent model of transient focal cerebral ischemia that matrix metalloproteinase-12 (MMP-12) induction in the ischemic brain promotes post-stroke blood-brain barrier disruption, apoptosis, demyelination, and infarction. The purpose of the present study is to investigate the role of elevated MMP-12 on post-stroke neurological function and to identify the time window of therapeutic opportunity for MMP-12 suppression. Methods: Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion and reperfusion. Cohorts of rats (n =8-15/group) were administered with either MMP-12 shRNA or scrambled shRNA sequence (vehicle control) expressing plasmids (1 mg/Kg; intravenous) formulated as nanoparticles. The differences in sample size of various cohorts were attributed to the exclusion criteria followed, high mortality rate in vehicle control-treated group, and sample size required for statistical analysis. To assess the reflex, balance, sensory, and motor functions, rats from various cohorts were subjected to modified neurological severity scoring (mNSS), adhesive removal test, beam walk test and rotarod test at day 1, 3 and 5 of reperfusion. To assess the time window of therapeutic opportunity, various cohorts of rats were treated at 5 min, 3h, and 6h of reperfusion. Investigators blinded to study groups analyzed all outcome parameters. Results: The post-stroke percent survival rate in cohorts treated with MMP12shRNA expressing plasmids range from 82 to 89 as compared to 67 in vehicle control-treated group. The cohort of rats treated at 5 min of reperfusion with MMP-12snRNA expressing plasmids showed significantly better functional recovery as assessed by various neurological tests. However, delayed administration of MMP-12snRNA expressing plasmid (either at 3h or 6h of reperfusion) failed to promote any significant improvement in post-stroke neurological recovery. Conclusions: Post-stroke induction of MMP-12 in the ischemic brain contributes to neurological deficits and impedes recovery. MMP-12 targeting treatments immediately after reperfusion could offer substantial therapeutic benefits.

Lymphatic filariasis (LF) affects 120 million people around the world and another 856 million people are at risk of acquiring the infection. Mass Drug Administration (MDA) spearheaded by the World Health Organization is the only current strategy to control this infection. Recent reports suggest that despite several rounds of MDA, elimination has not been achieved and there is a need for more stringent control strategies for control of LF. An effective prophylactic vaccine combined with MDA has significant potential. Initial trials using a prophylactic trivalent recombinant Brugia malayi heat shock protein 12.6, abundant larval transcript -2 and tetraspanin large extra-cellular loop (rBmHAT) vaccine developed in our laboratory conferred only 35% protection in macaques. Therefore, the focus of the present study was to improve the current vaccine formulation to obtain better protection in non-human primates. We made two modifications to the current formulation: (i) the addition of another antigen, thioredoxin peroxidase-2 (TPX-2) to make it a tetravalent vaccine (rBmHAXT) and (ii) the inclusion of an adjuvant; AL019 (alum plus glucopyranosyl lipid adjuvant-stable emulsion) that is known to promote a balanced Th1/Th2 response. A double-blinded vaccination trial was performed with 40 macaques that were divided into three treatment groups and one control group (n = 10/group). Vaccinated animals received 4 immunisations at 1 month intervals with 150 µg/ml of rBmHAT plus alum, rBmHAT plus AL019 or rBmHAXT plus AL019. Control animals received AL019 only. All vaccinated macaques developed significant (P ≤ 0.003) titers of antigen-specific IgG antibodies (1:20,000) compared with the controls. One month after the last dose, all macaques were challenged s.c. with 130–180 B. malayi L3s. Our results showed that seven out of 10 (70%) of macaques given the improved rBmHAXT vaccine did not develop the infection compared with AL019 controls, of which seven out of 10 macaques developed the infection. Titers of antigen-specific IgG1 and IgG2 antibodies were significantly (P ≤ 0.01) higher in vaccinated animals and there was an increase in the percentage of IL-4 and IFN-γ secreting antigen-responding memory T cells. These studies demonstrated that the improved formulation (rBmHAXT plus AL019) is a promising vaccine candidate against human lymphatic filariasis.

Mass drug administration (MDA) is the current strategy for interrupting the transmission of lymphatic filariasis (LF) infection and control of the disease in endemic areas. However, subject non-compliance has resulted in the presence of several "transmission hotspots" in the endemic regions threatening the reemergence of LF. This situation is further complicated by the fact that the drugs used in MDA are not effective against adult LF worms, a major concern for the control strategy. Thus, there is clearly a need for an effective and sustainable approach to control LF. Prophylactic vaccine combined with targeted treatment of infected patients and vector control is suggested as a more sustainable strategy to eliminate LF infection from endemic regions. A multivalent vaccine (rBmHAT) developed in our laboratory conferred about 90% protection in rodents. However, when we tested the rBmHAT vaccine along with alum in rhesus macaques, only about 40% protection was achieved and the immune response obtained was Th2 biased. In an attempt to improve the vaccine, in this study, we tested two vaccine antigens (rBmHAT and rBmHAX) along with two adjuvant formulations [alum + GLA (AL019) and mannosylated chitosan (MCA)] in a mouse model. Our results show that rBmHAT is a better vaccine antigen than rBmHAX. Combination of rBmHAT with AL019 or MCA adjuvants gave 94 and 88% protection, respectively, against challenge infections. Immunized animals developed antigen-specific memory T cells that secreted significant levels of IL-4, IFN-γ, and IL-17 suggesting the generation of a balanced Th1/Th2 responses following immunization. A major advantage of MCA adjuvant is that the vaccine booster doses can be administered orally. These studies thus showed that rBmHAT is a better vaccine antigen and can be given in combination with AL019 or MCA adjuvant to obtain excellent results.

According to the World Health Organization, currently, over 880 million people are at risk of acquiring the lymphatic filariasis (LF) infection in over 52 countries worldwide. Current approaches to control LF by 2020 are reaching short of its anticipated goal. Several studies suggest the existence of protective immunity against LF in the human. Thus, it is possible to develop a prophylactic vaccine against LF infections in the human. Several potential vaccine candidates were identified and tested for their vaccine potential against LF. Preclinical studies to date suggest that it is possible to develop a prophylactic vaccine against LF. Much work needs to be done, but it is clear that a prophylactic vaccine combined with targeted chemotherapy is critically required for eliminating LF worldwide.

A multivalent recombinant fusion protein prophylactic vaccine, rBmHAXT developed against lymphatic filariasis (LF) demonstrated over 57% protection against challenge infection in rhesus macaque model. Currently, we do not know if the rBmHAXT vaccination has any effect on adult worms and/or on the fecundity of adult female worms. Thus, the major focus of this study was to determine the effect of rBmHAXT vaccination on Brugia malayi infected mongolian gerbils. We performed two sets of experiments. In the first set of experiment, gerbils were infected with 100 B. malayi L3. After confirming the establishment of infection, four rounds of DEC treatment and rBmHAXT vaccination was given. Results showed that following vaccination with rBmHAXT, the microfilaria (Mf) count was significantly decreased in all vaccinated animals compared to controls. At the end of these experiments, we collected and counted the established adult worms. There was a 36% reduction in the recovery of adult female worms, which might account for the low Mf load in vaccinated animals. In the second set of experiments, animals were vaccinated first with rBmHAXT followed by surgically implanting adult male or female B. malayi parasites into the peritoneal cavity to determine the effect of vaccination on each sex of the parasite. Our results show that the rBmHAXT vaccination has no effect on male adult worms compared to controls. However, there was 40% reduction in the Mf load in vaccinated animals that were transplanted with adult female worms. These findings suggested that the rBmHAXT vaccination has potential damaging effect on the fecundity of adult female worms. Scanning electron microscopy studies showed cuticular damage on the surface of adult female worms. These studies thus show that the rBmHAXT vaccination in infected gerbils has partial microfilaricidal effect and potentially affect the fecundity of adult female worms.

In this study, we cloned the homologue of macrophage migration inhibitory factor (MIF) secreted by the human filarial parasite Wuchereria bancrofti (rWbaMIF-2) and evaluated its potential in ameliorating inflammation in a dextran sulfate sodium salt (DSS)-induced colitis model. DSS administration resulted in substantial loss of body weight along with bloody diarrhea, severe inflammation in the colon and reduced length of the colon in just under 7 days. Treatment with rWbaMIF-2 reversed the clinical symptoms in DSS administered mice, characterized by no blood in the stools, minimal inflammation on the colon wall and the mice maintained normal colon length. rWbaMIF-2 treatment downregulated TNF-α, IL-6, IFN-γ, IL-1β, IL-17A, and NOS2 genes in the colon tissue and upregulated the percentage of IL-10 producing Treg and B1 cells in the colon and peritoneal cavity. Thus, one of the mechanisms by which rWbaMIF-2 induce its anti-inflammatory effect appeared to be by promoting the infiltration of IL-10 producing Treg cells into the colon. Further analysis showed alternative activation of F4/80+ large peritoneal macrophages (LPMs) with increased expression of arginase (p≤0.05) and reduced expression of iNOS, MPO activity, TNF-α, IL-6 and TLR4 in LPMs following rWbaMIF-2 treatment. Thus, for the first time, we demonstrate here that rWbaMIF-2 treatment can activate LMP macrophages alternatively and promote infiltration of Treg cells into the colon to suppress inflammation in a colitis model. These studies also demonstrate the potential of developing rWbaMIF-2 as a small molecule therapeutic agent for colitis and other inflammatory conditions. Studies funded by NIH RO1 AI116441.

The therapeutic potential of filarial parasite derived recombinant cystatin was reported previously by our group in the mouse model of colitis. We showed that cystatin-treatment elevated IL-10+FoxP3+Tregs, IgM+B1a cells and AAMs in the colon and peritoneal cavity. In this study, we attempted to identify the therapeutically active peptide(s) of cystatin. Total length of cystatin is 157 aa. We first synthesized three 40-mer peptides of cystatin. 25μg of peptide 1, 2 or 3 was injected i.p. into groups of 10 mice each on days 3–6 following DSS-administration. Control mice received DMSO only. Mice that received whole cystatin protein remained as positive control. Following treatment, clinical and pathological disease parameters were determined. Our results show that treatment with peptide 1 and 2 significantly ameliorated the inflammatory responses in the colitis mice and significantly reversed the gross and histopathological changes in the colon. However, peptide 3 had no effect in alleviating the colitis symptoms. Analysis of the colon tissue showed that the myeloperoxidase activity was significantly (p

Lymphatic filariasis (LF) is a mosquito-transmitted tropical neglected parasitic infection that currently affects over 120 million people around the world and another 856 million people are at risk of acquiring the infection. Mass Drug Administration (MDA) spearheaded by the World Health Organization is the only current strategy to control this infection in endemic areas. In this study, we performed an epidemiological survey in select regions in the southern parts of India to determine the current status of LF infection in subjects. Night blood samples were collected from 916 subjects after proper consent and were screened for the presence of circulating microfilariae of Wuchereria bancrofti in their peripheral blood. Our results showed the presence of 51 (5.56%) cases of human LF infection in the surveyed areas including new cases for LF, which were not recorded previously. Given the presence of new cases of LF infections, we trapped mosquitoes from these regions and screened for the presence of W. bancrofti L3 specific Ssp1 DNA repeat sequences by PCR. Our results confirmed the presence of LF infection in the mosquitoes collected from six out of nine districts that we surveyed. These findings confirm active transmission of LF infection in all of the areas that we surveyed, despite several years of MDA treatment. The findings in this study suggest potential reemergence of LF infection in most of the areas we surveyed and warrants for a more stringent strategy for controlling LF in these endemic areas.

Lymphatic filariasis (LF) is a tropical parasitic infection of human transmitted by mosquitoes. Chronic infection results in severe physical disability in the infected patients. Although several potential vaccine antigens were identified by several groups, there are no licensed prophylactic vaccine to date against this infection in the human. Previous attempts from our laboratory to develop a trivalent prophylactic vaccine against LF showed that >90% protection could be achieved in rodent models. However, this trivalent vaccine gave only 35% protection in non-human primates. The major focus of this study was to develop a tetravalent prophylactic vaccine (rBmHAXT) and test the vaccine potential in a mouse model. We evaluated three different adjuvant formulations; alum, glucopyranosyl lipid adjuvant in stable emulsion (GLA/SE) alum (AL019), and mannosylated chitosan (MCA) to determine the optimum adjuvant formulation for rBmHAXT. Results presented in this study show that rBmHAXT + AL019 gave the highest rate of protection (>88%) against challenge infection, compared to rBmHAXT + AL007 (79%), rBmHAXT + MCA (79%) and controls. Analysis of the immune correlates of protection showed that all three adjuvants elicited high titer of antigen-specific IgG1, IgG2a, and IgG2b antibodies. High number of IFN-γ-producing antigen-specific memory cells were generated in the vaccinated animals irrespective of the adjuvants used. Similarly, spleen cells from rBmHAXT-vaccinated animals secreted IL-4, IL-10, and IFN-γ in response to rBmHAXT suggesting the generation of a balanced Th1/Th2 response. There was also an increase in IL-17-secreting cells in rBmHAXT-vaccinated animals. These findings thus suggest that rBmHAXT + AL019 is a better prophylactic formulation for LF.

Lymphatic filariasis (LF) is a mosquito-transmitted neglected tropical parasitic infection that causes most gruesome clinical outcome in the patients resulting in severe physical disability. Mass drug administration (MDA), spearheaded by the World Health Organization (WHO), is one of the most successful elimination strategies for any infection worldwide. India started implementing MDA since 2004 and has successfully reduced the incidence of the infection to a level that can be now easily managed. Few reports of non-compliance to MDA still remain. This has resulted in the presence of residual infections in some areas designated as “hotspots.” Transmission assessment surveys need to identify these “hotspots.” Before the momentum of MDA wanes, it is critically important to capitalize the success gained by MDA by planning toward an eradication strategy for LF in India. Eradication will not be possible with MDA alone. There is a need to develop a more sustainable strategy such as vaccine and vector control along with MDA. This paper reviews the current status of vaccine development against lymphatic filariasis and future prospects.

High-mobility group box 1 (HMGB1) is a dynamic nuclear protein participating in transcription, chromatin remodelling, and DNA recombination and repair processes. Accumulating evidence indicates that its function now extends beyond the nucleus, notably its extracellular role in inflammation. HMGB1 is implicated as a late mediator of sepsis and is also believed to promote atherosclerosis and other inflammatory diseases such as rheumatoid arthritis and systemic lupus erythematosus. Interestingly, deregulation of HMGB1 is shown to be associated with the hallmarks of cancer development. Moreover, several clinical studies have shown that HMGB1 is a promising biomarker for a variety of cancer types. In this review, we provide novel insights into the role and mechanisms of HMGB1, in particular, to hormone-related cancers and its potential to serve as a therapeutic target.

Lymphatic filariasis affects approximately 3% of the whole world population. Mass drug administration is currently the major control strategy to eradicate this infection from endemic regions by year 2020. Combination drug treatments are highly efficient in controlling the infection. However, there are no effective vaccines available for human or animal lymphatic filariasis despite the identification of several subunit vaccines. Lymphatic filariasis parasites are multicellular organisms and potentially use multiple mechanisms to survive in the host. Therefore, there is a need to combine two or more vaccine candidate antigens to achieve the desired effect. In this study we combined three well characterized vaccine antigens of Brugia malayi , heat shock protein 12.6 (HSP12.6), Abundant Larval transcript-2 (ALT-2) and tetraspanin large extra cellular loop (TSP-LEL) as a multivalent fusion vaccine. Putative immune individuals carry circulating antibodies against all three antigens. Depletion of these antigen specific antibodies from the sera samples removed the ability of the sera to participate in the killing of B. malayi L3 in an antibody dependent cellular cytotoxicity (ADCC) mechanism. Vaccination trials in mice with a bivalent [HSP12.6 + ALT-2 (HA), HSP12.6 + TSP-LEL (HT) or TSP-LEL + ALT-2 (TA)] or trivalent [HSP12.6 + ALT-2 + TSP-LEL (HAT)] vaccines using DNA, protein or heterologous prime boost regimen showed that trivalent HAT vaccine either as protein alone or as heterologous prime boost vaccine could confer significant protection (95%) against B. malayi L3 challenge. Immune correlates of protection suggest a Th1/Th2 bias. These finding suggests that the trivalent HAT fusion protein is a promising prophylactic vaccine against lymphatic filariasis infection in human.

Toxoplasma gondii is the causative agent for toxoplasmosis. The rhoptry protein 1 (ROP1) is secreted by rhoptry, an apical secretory organelle of the parasite. ROP1 plays an important role in host cell invasion. In this study, the efficacy of ROP1 as a vaccine candidate against toxoplasmosis was evaluated through intramuscular or subcutaneous injection of BALB/c mice followed by immunological characterization (humoral- and cellular-mediated) and lethal challenge against virulent T. gondii RH strain in BALB/c mice. Briefly, a recombinant DNA plasmid (pVAX1-GFP-ROP1) was expressed in CHO cells while expression of recombinant ROP1 protein (rROP1) was carried out in Escherichia coli expression system. Immunization study involved injection of the recombinant pVAX1-ROP1 and purified rROP1 into different group of mice. Empty vector and PBS served as two different types of negative controls. Results obtained demonstrated that ROP1 is an immunogenic antigen that induced humoral immune response whereby detection of a protein band with expected size of 43 kDa was observed against vaccinated mice sera through western blot analysis. ROP1 antigen was shown to elicit cellular-mediated immunity as well whereby stimulated splenocytes with total lysate antigen (TLA) and rROP1 from pVAX1-ROP1 and rROP1-immunized mice, respectively, readily proliferated and secreted large amount of IFN-γ (712 ± 28.1 pg/ml and 1457 ± 31.19 pg/ml, respectively) and relatively low IL-4 level (94 ± 14.5 pg/ml and 186 ± 14.17 pg/ml, respectively). These phenomena suggested that Th1-favored immunity was being induced. Vaccination with ROP1 antigen was able to provide partial protection in the vaccinated mice against lethal challenge with virulent RH strain of tachyzoites. These findings proposed that the ROP1 antigen is a potential candidate for the development of vaccine against toxoplasmosis.

Antibodies are widely available and cost-effective research tools in life science, and antibody conjugates are now extensively used for targeted therapy, immunohistochemical staining, or in vivo diagnostic imaging of cancer. Significant advances in site-specific antibody labeling technologies have enabled the production of highly characterized and homogenous conjugates for biomedical purposes, and some recent studies have utilized site-specific labeling to synthesize bifunctional antibody conjugates with both imaging and drug delivery properties. While these advances are important for the clinical safety and efficacy of such biologics, these techniques can also be difficult, expensive, and time-consuming. Furthermore, antibody-drug conjugates (ADCs) used for tumor treatment generally remain distinct from conjugates used for diagnosis. Thus, there exists a need to develop simple dual-labeling methods for efficient therapeutic and diagnostic evaluation of antibody conjugates in pre-clinical model systems. Here, we present a rapid and simple method utilizing commercially available reagents for synthesizing a dual-labeled fluorescent ADC. Further, we demonstrate the fluorescent ADC’s utility for simultaneous targeted therapy and molecular imaging of cancer both in vitro and in vivo. Employing non-site-specific, amine-reactive chemistry, our novel biopharmaceutical theranostic is a monoclonal antibody specific for a carcinoembryonic antigen (CEA) biomarker conjugated to both paclitaxel and a near-infrared (NIR), polyethylene glycol modified (PEGylated) fluorophore (DyLight™ 680-4xPEG). Using in vitro systems, we demonstrate that this fluorescent ADC selectively binds a CEA-positive pancreatic cancer cell line (BxPC-3) in immunofluorescent staining and flow cytometry, exhibits efficient internalization kinetics, and is cytotoxic. Model studies using a xenograft of BxPC-3 cells in athymic mice also show the fluorescent ADC’s efficacy in detecting tumors in vivo and inhibiting tumor growth more effectively than equimolar amounts of unconjugated drug. Overall, our results demonstrate that non-selective, amine-targeting chemistry is an effective dual-labeling method for synthesizing and evaluating a bifunctional fluorescent antibody-drug conjugate, allowing concurrent detection, monitoring and treatment of cancer.

During the last one decade, the whole world witnessed one of the most coordinated efforts toward global elimination of lymphatic filariasis (LF), a neglected tropical parasitic infection that affects 120 million people living in 72 different tropical countries. The approach was to use annual mass drug administration (MDA) using a combination of two chemotherapeutic agents to clear circulating parasites (microfilaria) in infected individuals living in various endemic regions of the world. This approach substantially decreased the incidence of infection in almost all the countries where the program was initiated. However, the biggest challenge now is to sustain the success and attain prophylaxis. This can be achieved only by newer chemotherapeutic agents against adult worms and an effective vaccine that can prevent future infections. This chapter summarizes the current status of LF elimination and the need for a more stringent and sustainable approach to control LF infection in endemic regions.

Lymphatic filariasis (LF) affects 120 million people around the world and another 856 million people are at risk of acquiring the infection. Mass Drug Administration (MDA) spearheaded by the World Health Organization is the only current strategy to control this infection. Recent reports suggest that despite several rounds of MDA, elimination has not been achieved and there is a need for more stringent control strategies for control of LF. An effective prophylactic vaccine combined with MDA has significant potential. Initial trials using a prophylactic trivalent recombinant Brugia malayi heat shock protein 12.6, abundant larval transcript -2 and tetraspanin large extra-cellular loop (rBmHAT) vaccine developed in our laboratory conferred only 35% protection in macaques. Therefore, the focus of the present study was to improve the current vaccine formulation to obtain better protection in non-human primates. We made two modifications to the current formulation: (i) the addition of another antigen, thioredoxin peroxidase-2 (TPX-2) to make it a tetravalent vaccine (rBmHAXT) and (ii) the inclusion of an adjuvant; AL019 (alum plus glucopyranosyl lipid adjuvant-stable emulsion) that is known to promote a balanced Th1/Th2 response. A double-blinded vaccination trial was performed with 40 macaques that were divided into three treatment groups and one control group (n = 10/group). Vaccinated animals received 4 immunisations at 1 month intervals with 150 µg/ml of rBmHAT plus alum, rBmHAT plus AL019 or rBmHAXT plus AL019. Control animals received AL019 only. All vaccinated macaques developed significant (P ≤ 0.003) titers of antigen-specific IgG antibodies (1:20,000) compared with the controls. One month after the last dose, all macaques were challenged s.c. with 130-180 B. malayi L3s. Our results showed that seven out of 10 (70%) of macaques given the improved rBmHAXT vaccine did not develop the infection compared with AL019 controls, of which seven out of 10 macaques developed the infection. Titers of antigen-specific IgG1 and IgG2 antibodies were significantly (P ≤ 0.01) higher in vaccinated animals and there was an increase in the percentage of IL-4 and IFN-γ secreting antigen-responding memory T cells. These studies demonstrated that the improved formulation (rBmHAXT plus AL019) is a promising vaccine candidate against human lymphatic filariasis.

Yersinia pestisis the etiologic agent of bubonic and pneumonic plagues. It is speculated thatY. pestishijacks antigen-presenting cells (APCs), such as dendritic cells (DCs) and alveolar macrophages, in order to be delivered to lymph nodes. However, how APCs initially capture the bacterium remains uncharacterized. It is well known that HIV-1 uses human DC-specific intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN) (CD209) receptor, expressed by APCs, to be captured and delivered to target cell, such as CD4+lymphocytes. Several gram-negative bacteria utilize their core lipopolysaccharides (LPS) as ligands to interact with the human DC-SIGN. Therefore, it is possible thatY. pestis, whose core LPS is naturally exposed, might exploit DC-SIGN to invade APCs. We demonstrate in this study thatY. pestisdirectly interacts with DC-SIGN and invades both DCs and alveolar macrophages. In contrast, when engineered to cover the core LPS,Y. pestisloses its ability to invade DCs, alveolar macrophages, and DC-SIGN-expressing transfectants. The interaction betweenY. pestisand human DCs can be reduced by a combination treatment with anti-CD209 and anti-CD207 antibodies. This study shows that human DC-SIGN is a receptor forY. pestisthat promotes phagocytosis by DCs in vitro.

A cloning and expression system that allows display of proteins on the surface of filamentous phages was exploited to display a 28-kDa glutathione S -transferase (Sm28GST) antigen of the human parasite Schistosoma mansoni . The phage-displayed Sm28GST (pdGST) was immunoreactive and was recognized by immune sera, suggesting that the Sm28GST protein displayed on the surface of phages potentially maintains native conformation. Subsequent immunization studies showed that mice can develop high titers of antibodies against pdGST and do not require any additional adjuvant for immunization. Isotype analysis suggested that the pdGST immunization predominantly induced immunoglobulin G2b (IgG2b), IgG3, and IgM anti-GST antibodies in mice. Furthermore, the pdGST immunization was found to confer about 30% protection after a challenge infection with 100 cercariae of S. mansoni in BALB/c mice. These findings suggest that phage display is a simple, efficient, and promising tool to express candidate vaccine antigens for immunization against infectious agents.

Background: Toxoplasma gondii infects all warm-blooded animals, including humans. Early diagnosis and determining the infective stage are critical for effectively treating immunosuppressed individuals and pregnant women with toxoplasmosis. Among the rhoptry proteins of the parasite, Rhoptry protein 8 (ROP8), is known to be expressed during the early stages of T. gondii infection and is involved in parasitophorous vacuole formation. In this study, we have investigated the diagnostic efficacy of recombinant ROP8 (rROP8). Methods: The ROP8 gene was cloned into pCOLD ID NA vector andexpressed as a soluble recombinant antigen in Escherichia coli. Expressed ROP8 protein was evaluated using western blot method. Results: Western blot analysis of purified rROP8 antigen using 200 T. gondii-infected human serum samples, as well as non-infected serum controls, allowed for the successful identification of toxoplasmosis-positives, yielding a 90% sensitivity and 94% specificity. Conclusion: Our findings indicated that rROP8 antigen expressed in E. coli was able to detect toxoplasmosis in infected human serum with specificity and sensitivity suggesting that rROP8 antigen represents a valid diagnostic marker for toxoplasmosis.

Developing an effective vaccine against lymphatic filariasis will complement the WHO's effort to eradicate the infection from endemic areas. Currently 83 different countries are endemic for this infection and over 1 billion people are at risk. An effective vaccine coupled with mass drug administration will reduce the morbidity and social stigma associated with this gruesome disease. Several potential vaccine candidates that can confer partial protection in experimental animals have been reported from different laboratories. However, no licensed vaccines are currently available for this disease. Among the several vaccine antigens identified from our laboratory, three most promising antigens; rBmHSPαc (α crystalline domain and c-terminal extension of Heat Shock Protein 12.6), rBmALT-2 (Abundant larval transcript) and rBmTSP LEL (Tetraspanin large extracellular loop) was further developed as a recombinant fusion protein vaccine (rBmHATαc). In a mouse model this fusion protein vaccine gave close to 68% protection following a challenge infection. To improve the vaccine efficiency of rBmHATαc, in this study we evaluated various preparations of alum (AL007, AL019, Alhydrogel and Imject® Alum) as adjuvants. Our results show that mice immunized with rBmHATαc formulated in AL007 (alum from IDRI) and/or AL019 (alum plus TLR-4 agonist from IDRI) gave the highest IgG antibody titer compared to other groups. Subsequent in vivo challenge experiments confirmed that >95% protection can be achieved when AL007 or AL019 was used as the adjuvant. However, when Imject® Alum or alhydrogel was used as the adjuvant only 76% and 72% protection respectively could be achieved. These results show that AL007 or AL019 (IDRI) is an excellent choice of adjuvant for the rBmHATαc vaccine against B. malayi L3 in mice.