Evaluation of Wuchereria bancrofti GST as a Vaccine Candidate for Lymphatic Filariasis

Lymphatic parasites survive for years in a complex immune environment by adopting various strategies of immune modulation, which includes counteracting the oxidative free radical damage caused by the host. We now know that the filarial parasites secrete antioxidant enzymes. Among these, the glutathione-S-transferases (GSTs) have the potent ability to effectively neutralize cytotoxic products arising from reactive oxygen species (ROS) that attack cell membranes. Thus, GSTs have the potential to protect the parasite against host oxidative stress. GSTs of several helminthes, including schistosomes, fasciola and the filarial parasite Seteria cervi, are also involved in inducing protective immunity in the host. The schistosome 28 kDa GST has been successfully developed into a vaccine and is currently in Phase II clinical trials. Thus, GST appears to be a potential target for vaccine development. Therefore, in the present study, we cloned W. bancrofti GST, and expressed and purified the recombinant protein. Immunization and challenge experiments showed that 61% of protection could be achieved against B. malayi infections in a jird model. In vitro studies confirm that the anti-WbGST antibodies participate in the killing of B. malayi L3 through an ADCC mechanism and enzymatic activity of WbGST appears to be critical for this larvicidal function

Biochemical Characterization and Evaluation of a Brugia malayi Small Heat Shock Protein as a Vaccine against Lymphatic Filariasis

Filarial nematodes enjoy one of the longest life spans of any human pathogen due to effective immune evasion strategies developed by the parasite. Among the various immune evasion strategies exhibited by the parasite, Interleukin 10 (IL-10) productions and IL-10 mediated immune suppression has significant negative impact on the host immune system. Recently, we identified a small heat shock protein expressed by Brugia malayi (BmHsp12.6) that can bind to soluble human IL-10 receptor alpha (IL-10R) and activate IL-10 mediated effects in cell lines. In this study we show that the IL-10R binding region of BmHsp12.6 is localized to its N-terminal region. This region has significant sequence similarity to the receptor binding region of human IL-10. In vitro studies confirm that the N-terminal region of BmHsp12.6 (N-BmHsp12.6) has IL-10 like activity and the region containing the alpha crystalline domain and C-terminus of BmHsp12.6 (BmHsp12.6αc) has no IL-10 like activity. However, BmHsp12.6αc contains B cell, T cell and CTL epitopes. Members of the sHSP families are excellent vaccine candidates. Evaluation of sera samples from putatively immune endemic normal (EN) subjects showed IgG1 and IgG3 antibodies against BmHsp12.6αc and these antibodies were involved in the ADCC mediated protection. Subsequent vaccination trials with BmHsp12.6αc in a mouse model using a heterologous prime boost approach showed that 83% protection can be achieved against B. malayi L3 challenge. Results presented in this study thus show that the N-BmHsp12.6 subunit of BmHsp12.6 has immunoregulatory function, whereas, the BmHsp12.6αc subunit of BmHsp12.6 has significant vaccine potential

Pulmonary inflammation and immune responses to the namatode nippostrongylus brasiliensis

Bibliography: p. 294-358

Sumoylation of Human Translationally Controlled Tumor Protein Is Important for Its Nuclear Transport

Translationally controlled tumor protein (TCTP) lacks nuclear bipartite localization signal sequence; yet TCTP is present abundantly in the nucleus. At present it is not known how TCTP gets transported to the nucleus. Sequence analyses showed that all TCTPs described to date have putative small ubiquitin-like modifier (SUMO) motifs. Since SUMO modification plays an important role in the nuclear transport of proteins, we evaluated whether SUMO motifs are important for transport of TCTP into the nucleus. We show that TCTP exists in sumoylated form in cytoplasm and nucleus of mammalian cells. Point mutation of lysine residue in the SUMO motif compromised the ability of TCTP to get sumoylated in vitro. When cells were transfected with FLAG-tagged mutated TCTP, nuclear transport of TCTP was inhibited confirming that sumoylation is critical for the nuclear transport of TCTP. Our previous studies demonstrated that TCTP can function as an antioxidant protein in the nucleus. When we mutated TCTP at the SUMO motif the antioxidant function of TCTP was compromised. Results presented in this study thus show that sumoylation plays an important role in the transport of TCTP into the nucleus where they function as antioxidant protein

Sumoylation of Human Translationally Controlled Tumor Protein Is Important for Its Nuclear Transport

Translationally controlled tumor protein (TCTP) lacks nuclear bipartite localization signal sequence; yet TCTP is present abundantly in the nucleus. At present it is not known how TCTP gets transported to the nucleus. Sequence analyses showed that all TCTPs described to date have putative small ubiquitin-like modifier (SUMO) motifs. Since SUMO modification plays an important role in the nuclear transport of proteins, we evaluated whether SUMO motifs are important for transport of TCTP into the nucleus. We show that TCTP exists in sumoylated form in cytoplasm and nucleus of mammalian cells. Point mutation of lysine residue in the SUMO motif compromised the ability of TCTP to get sumoylated in vitro. When cells were transfected with FLAG-tagged mutated TCTP, nuclear transport of TCTP was inhibited confirming that sumoylation is critical for the nuclear transport of TCTP. Our previous studies demonstrated that TCTP can function as an antioxidant protein in the nucleus.When we mutated TCTP at the SUMO motif the antioxidant function of TCTP was compromised. Results presented in this study thus show that sumoylation plays an important role in the transport of TCTP into the nucleus where they function as antioxidant protein

Multivalent Vaccine Formulation with BmVAL-1 and BmALT-2 Confer Significant Protection against Challenge Infections with Brugia malayi in Mice and Jirds

Purpose: Lymphatic filariasis is a mosquito borne infection affecting 120 million people in 83 different countries. Despite several setbacks, mass drug administration is fully underway in several parts of the world to eradicate this infection by year 2020. Even though drug alone is highly efficient in treating this infection, long term sustainable prophylaxis needs effective vaccine. Unfortunately there are no vaccines available to control this infection in human and animals despite the fact that several potential candidate vaccine antigens have been identified by several laboratories. Brugia malayi Vespid venom Allergen homologue-Like protein (BmVAL-1) and B. malayi Abundant Larval Transcript (BmALT-2) are two of the most promising vaccine candidates. In this study we have evaluated various vaccination regimens consisting of DNA and protein antigens and evaluated the potential of monovalent and multivalent vaccine formulations in mice and jird animal models. Methods: Mice and jirds were vaccinated with monovalent DNA preparations of BmVAL-1 or BmALT-2 in pVAX-1 vector or monovalent protein preparations of rBmVAL-1 and rBmALT-2 in alum using a homologous or heterologous prime boost approach. These vaccine regimens were then compared with a multivalent vaccine formulation consisting of DNA or hybrid protein formulation of the two antigens. Challenge experiments were performed with B. malayi L3 in mice and jirds to evaluate the degree of protection and immunological parameters were determined in mice and human to elucidate the characteristics of the protective immune responses. Results: Results presented in this study show that vaccination with monovalent BmVAL-1 vaccine confers from 39% (DNA vaccine) protection to 54% (DNA prime and protein boost) protection in mice. Similar degree of protection was observed in jirds (50% to 52% protection). Monovalent BmAT-2 afforded 51% to 75% protection in mice and 58% to 79% protection in jirds. When we tested a multivalent formulation of BmVAL-1 and BmALT-2, there was 57% to 82% protection in mice and 77% to 85% protection in jirds. Heterologous prime boost approach using the multivalent vaccine gave the highest degree of protection in both mice and jirds. Serological analysis in mice showed that BmVAL-1 vaccination induced an IgG1, IgG2a and IgG3 antibody response, whereas, BmALT-2 vaccination predominantly induced an IgG1 and IgG3 antibody response. Cytokine responses of antigen responding cells in the spleen secreted predominantly IFN-y and IL-5 in response to BmVAL-1 and IL-4 and IL-5 in response to BmALT-2. Conclusion: In conclusion, results presented in this study show that a multivalent vaccine formulation of BmVAL-1 and BmALT-2 when given as a prime boost regimen gave significant protection against lymphatic filariasis caused by B. malayi in mice and jirds. Since putatively immune EN subjects also carry protective antibodies against BmVAL-1 and BmALT-2, there is a great potential for developing this multivalent formulation as a prophylactic vaccine against B. malayi for human and veterinary use

Evaluating the Vaccine Potential of a Tetravalent Fusion Protein (rBmHAXT) Vaccine Antigen Against Lymphatic Filariasis in a Mouse Model

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

Expression of a 28-Kilodalton Glutathione S-Transferase Antigen of Schistosoma mansoni on the Surface of Filamentous Phages and Evaluation of Its Vaccine Potential

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