Abundant Larval Transcript Research Articles

Bacterial lipid modification enhances immunoprophylaxis of filarial abundant larval transcript‐2 protein in Mastomys model

As in many other parasitic diseases, efficacious vaccine for lymphatic filariasis has been elusive for want of new approaches leaving billions of people either debilitated or at risk. With multiple B- and T-cell epitopes, the abundant larval transcript-2 (ALT-2) of the filarial worm, Brugia malayi, has been shown to be a promising immunoprophylactic target. To enhance its efficacy, it was lipid modified using our recently developed protein engineering tool, which then offered 30% more immunoprotection (49 vs. 79%) in Mastomys coucha model. Sustained high levels of IFN-γ (about 100 times) and high antibody titres (10-fold) elicited by lipid-modified ALT-2, as compared to the native form, indicated the maintenance of Th1/Th2 balance that is impaired in filariasis. Thus, this study provides the basis for developing efficacious vaccines for filariasis and other parasitic diseases by exploiting bacterial lipid modification.

Nematode-Derived Proteins Suppress Proliferation and Cytokine Production of Antigen-Specific T Cells via Induction of Cell Death

In order to establish long-lasting infections in their mammalian host, filarial nematodes have developed sophisticated strategies to dampen their host’s immune response. Proteins that are actively secreted by the parasites have been shown to induce the expansion of regulatory T cells and to directly interfere with effector T cell function. Here, we analyze the suppressive capacity of Onchocerca volvulus -derived excreted/secreted proteins. Addition of two recombinant O . volvulus proteins, abundant larval transcript-2 (OvALT-2) and novel larval transcript-1 (OvNLT-1) to cell cultures of T cell receptor transgenic CD4+ and CD8+ T cells suppressed antigen-specific stimulation in vitro. Ovalbumin-specific CD4+ DO11.10 and OT-II T cells that had been stimulated with their cognate antigen in the presence of OvALT-2 or OvNLT-1 displayed reduced DNA synthesis quantified by 3H-thymidine incorporation and reduced cell division quantified by CFSE dilution. Furthermore, the IL-2 and IFN-γ response of ovalbumin-specific CD8+ OT-I T cells was suppressed by OvALT-2 and OvNLT-1. In contrast, another recombinant O . volvulus protein, microfilariae surface-associated antigen (Ov103), did not modulate T cell activation, thus serving as internal control for non-ESP-mediated artifacts. Suppressive capacity of the identified ESP was associated with induction of apoptosis in T cells demonstrated by increased exposure of phosphatidylserine on the plasma membrane. Of note, the digestion of recombinant proteins with proteinase K did not abolish the suppression of antigen-specific proliferation although the suppressive capacity of the identified excreted/secreted products was not mediated by low molecular weight contaminants in the undigested preparations. In summary, we identified two suppressive excreted/secreted products from O . volvulus , which interfere with the function of antigen-specific T cells in vitro.

Single multivalent vaccination boosted by trickle larval infection confers protection against experimental lymphatic filariasis

The multivalent vaccine BmHAT, consisting of the Brugia malayi infective larval (L3) antigens heat shock protein12.6 (HSP12.6), abundant larval transcript-2 (ALT-2) and tetraspanin large extra cellular loop (TSP-LEL), was shown to be protective in rodent models from our laboratory. We hypothesize that since these antigens were identified using protective antibodies from immune endemic normal individuals, the multivalent vaccine can be augmented by natural L3 infections providing protection to the vaccinated host. This hypothesis was tested using single dose of DNA and protein or protein alone of the BmHAT vaccination in gerbils followed by live trickle L3 infection as booster dose. Vaccine-induced protection in gerbils was determined by worm establishment, micropore chamber assay and by antibody dependant cell cytotoxicity (ADCC) assay. Results were compared with the traditional prime-boost vaccination regimen. Gerbils vaccinated with BmHAT and boosted with L3 trickle infection were protected 51% (BmHAT DNA–protein) and 48% (BmHAT protein) respectively. BmHAT vaccination plus L3 trickle booster generated significant titer of antigen-specific IgG antibodies comparable to the traditional prime boost vaccination approach. BmHAT vaccination plus L3 trickle booster also generated antigen-specific cells in the spleen of vaccinated animals and these cells secreted predominantly IFN-γ and IL-4 in response to the vaccine antigens. These studies thus show that single dose of BmHAT multivalent vaccination followed by L3 trickle booster infection can confer significant protection against lymphatic filariasis.

Multivalent fusion protein vaccine for lymphatic filariasis

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.

Protective immune responses to biolistic DNA vaccination of Brugia malayi abundant larval transcript-2

Biolistic vaccination using gene gun is developed as a safer tool for delivery of DNA vaccines, a technique that combines high vaccine efficiency with lower antigen dosage and lower cost per vaccine dose. In this study, we compared the protective responses in mice after delivering the Brugia malayi abundant larval transcript-2 (BmALT-2) DNA vaccine using the conventional intradermal approach or with the needleless gene gun delivery approach. BmALT-2 is a leading vaccine candidate against B. malayi, a lymphatic filarial parasite of human. After optimizing the DNA dose and gene gun parameters for delivery into mouse skin, groups of mice were biolistically vaccinated with 5μg of BmALT-2pVAX. Groups of mice vaccinated intradermally with 5μg or 100μg of BmALT-2pVAX was used for comparison of vaccine efficacy. Results demonstrated that gene gun vaccination with 5μg of BmALT-2pVAX conferred significant protection against challenge infection that was comparable to the degree of protection conferred by intradermal vaccination with 100μg of BmALT-2pVAX. This observation was further supported by an in vitro antibody dependent cellular cytotoxicity (ADCC) assay. Analysis of the immune response showed that the gene gun vaccination predominantly induced an IgG1 antibody response and significantly high Th2 cytokine response (IL-4) from spleen cells compared to intradermal BmALT-2 DNA delivery that induced predominantly an IgG2a and Th1 cytokine response (IFN-γ, IL-12 and TNF-α). These findings show that host protective responses could be achieved with 20 fold decrease in DNA dose using a gene gun and could prove to be an efficient delivery method in BmALT-2 DNA vaccination against lymphatic filariasis.

The adjuvant-free immunoprotection of recombinant filarial protein Abundant Larval Transcript-2 (ALT-2) in Mastomys coucha and the immunoprophylactic importance of its putative signal sequence

The filarial protein Abundant Larval Transcript-2 (ALT-2) of the filarial parasite Brugia malayi has been shown to produce 74% worm clearance when administered with an adjuvant. In the present study, we show that it not only induces humoral and cell-mediated immunity, but also protection up to 71% in Mastomys coucha, a permissive animal model for filariasis, even without adjuvant. This unique feature of ALT-2 protein is highly restricted to its 21 amino acid N-terminal signal sequence, the absence of which resulted in poor immune response as well as immunoprotection (49%). Moreover, ALT-2 is likely to exert immunoprotection effects in B. malayi infection by maintaining a Th1–Th2 balance, evident from higher levels of IgG1 and IgG2a as well as IL-4 and IFN-γ. An improved understanding about the role of this filarial protein in host immunity, host–parasite interaction and worm clearance will aid in the development of good immunoprophylaxis for the disease.

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, a mosquito-borne infection, affects 120 million people in 83 different countries. Mass drug administration is fully underway in several parts of the world to eradicate this infection by year 2020. Drugs alone are highly efficient treatments, but long-term sustainable prophylaxis requires an effective vaccine. No vaccines are available for humans and animals despite several potential candidate vaccine antigens having been identified. Brugia malayi vespid venom allergen homolog-like protein (BmVAL-1) and B. malayi abundant larval transcript (BmALT-2) are two of the most promising vaccine candidates. We 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 humans to elucidate the characteristics of the protective immune responses. RESULTS: Vaccination with monovalent BmVAL-1 vaccine conferred 39% (DNA vaccine) to 54% (DNA prime and protein boost) protection in mice. A similar degree of protection was observed in jirds (50% to 52%). Monovalent BmALT-2 afforded 51% to 75% protection in mice and 58% to 79% protection in jirds. Our testing of a multivalent formulation of BmVAL-1 and BmALT-2, showed 57% to 82% protection in mice and 77% to 85% protection in jirds. A 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-γ and IL-5 in response to BmVAL-1, and IL-4, and IL-5 in response to BmALT-2. CONCLUSION: A multivalent vaccine formulation of BmVAL-1 and BmALT-2 given as a prime boost regimen gave significant protection against lymphatic filariasis caused by B. malayi in mice and jirds. Because putatively immune endemic normal subjects also carry protective antibodies against BmVAL-1 and BmALT-2, developing this multivalent formulation as a prophylactic vaccine against B. malayi for human and veterinary use has great potential.

Crucial epitopes of Wuchereria bancrofti abundant larval transcript recognized in natural infection

Lymphatic filariasis is a tropical disease, with over 40 million people seriously incapacitated due to lymphangitis and elephantiasis. Over 99% of infections are caused by the nematode Wuchereria bancrofti. Expressed sequence tag (EST) analysis of filarial genome identified novel infective larval (L3) stage-specific antigen, abundant larval transcript (ALT-2), which was shown to be highly essential for parasite establishment and survival in the host. The unique structural features and immunological characteristics of ALT-2 indicate the presence of critical motifs that needs to be explored in natural human infection for understanding and management of the disease. In order to dissect the epitopes of ALT protein recognized in humans, eight peptides spanning the entire protein sequence were selected based on in-silico epitope prediction and synthesized. Analysis of the reactivity of W. bancrofti ALT-2 synthetic peptides with clinical sera (n = 40) from endemic areas identified epitopes recognized by putatively immune sera, of which two comprise the highly variable acidic domain (21-60). Interestingly, our study also revealed crucial epitopes recognized by microfilaremic (MF) sera with significantly high IgG4 isotype (p < 0.001), implicated in immunomodulation. The epitope peptides identified were highly specific for MF sera and, thus, have the potential to be exploited as diagnostic markers.

Production and single-step purification of Brugia malayi abundant larval transcript (ALT-2) using hydrophobic interaction chromatography

Abundant larval transcript (ALT), a novel filarial protein, has been shown to have great potential as a vaccine in the prevention of human lymphatic filariasis. In this study, we report a method for the production of recombinant ALT-2 protein, expressed in the cytoplasm of bacterium Escherichia coli in soluble form and purification in a single step using hydrophobic interaction chromatography (HIC). Fermentation was done by continuous fed-batch methodology with dissolved oxygen (DO)-controlled feed addition. The culture was induced with 1mM isopropyl-β-D: -thiogalactopyranoside (IPTG). Up to 9g/l dry cell weight (DCW) of biomass was obtained from 1.6l of Luria-Bertani (LB) broth in a bench-scale reactor. Around 200mg/l of purified ALT-2 with a yield of about 60% was obtained. This is almost a 2.5-fold increase in final protein yield compared to purification using immobilized metal affinity chromatography (IMAC).

Lymphatic filarial species differentiation using evolutionarily modified tandem repeats: Generation of new genetic markers

Polymerase chain reaction based methods are promising tools for the monitoring and evaluation of the Global Program for the Elimination of Lymphatic Filariasis. The currently available PCR methods do not differentiate the DNA of Wuchereria bancrofti or Brugia malayi by a single PCR and hence are cumbersome. Therefore, we designed a single step PCR strategy for differentiating Bancroftian infection from Brugian infection based on a newly identified gene from the W. bancrofti genome, abundant larval transcript-2 (alt-2), which is abundantly expressed. The difference in PCR product sizes generated from the presence or absence of evolutionarily altered tandem repeats in alt-2 intron-3 differentiated W. bancrofti from B. malayi. The analysis was performed on the genomic DNA of microfilariae from a number of patient blood samples or microfilariae positive slides from different Indian geographical regions. The assay gave consistent results, differentiating the two filarial parasite species accurately. This alt-2 intron-3 based PCR assay can be a potential tool for the diagnosis and differentiation of co-infections by lymphatic filarial parasites.

Evaluation of Immunoprophylactic Efficacy of Brugia malayi Transglutaminase (BmTGA) in Single and Multiple Antigen Vaccination with BmALT-2 and BmTPX for Human Lymphatic Filariasis

An attempt was made to study the immunoprophylactic efficacy of recombinant Brugia malayi transglutaminase (BmTGA) as protein vaccine along with two other recombinant proteins, Brugia malayi abundant larval transcript-2 (BmALT-2) and Brugia malayi thioredoxin peroxidase (BmTPX), in single and multiple antigen form for human lymphatic filariasis. Parasite challenge studies in jirds exhibited protection of 30%, 69%, and 43% against BmTGA, BmALT-2, and BmTPX, respectively, in single antigen vaccination mode. The protective efficacy of BmTGA was enhanced significantly (74%) by immunizing the jirds in multiple antigen vaccination mode along with BmTPX, whereas immunizing with the combination of BmTGA and BmALT2 conferred only 47% protection. The same protection profiles were obtained by in vitro antibody-dependent cellular cytotoxicity, using live microfilariae and L3 stage larvae. The immune response was Th2 biased, irrespective of single or multiple vaccinations. The combination of BmTGA and BmTPX seems to be a promising vaccine candidate against lymphatic filariasis.

Comparison of immunogenicity, protective efficacy of single and cocktail DNA vaccine of Brugia malayi abundant larval transcript (ALT-2) and thioredoxin peroxidase (TPX) in mice

Although DNA vaccines have several advantages over conventional vaccines, antibody production and protection are often not adequate, particularly in single plasmid vaccine formulation. In the present study we evaluated the efficacy of a cocktail vaccine based on plasmids encoding larval (L3) stage-specific Brugia malayi abundant larval transcript (BmALT-2) and antioxidant detoxification enzyme B. malayi thioredoxin peroxidase (BmTPX) to induce antibodies, protective efficacy and cell-mediated immune response in mice. Mice immunized with cocktail DNA vaccines containing the pVAX ALT-2 + TPX developed higher titers of anti-BmALT-2 + TPX (1:5000) antibodies, compared to the mice immunized with single DNA vaccine of pVAX ALT-2 or pVAX TPX (1:2000). Correlating with this, the mice administered with cocktail vaccine induced up to 78% of cytotoxicity against B. malayi mf. This cytotoxicity was high compared to 34% induced by the pVAX-ALT2 or 37% by pVAX-TPX. Moreover, cocktail vaccination of mice resulted in significantly higher level of cellular proliferative response associated with raised levels of IFN-γ that skewed towards Th1 type of response compared to vaccination using either of the components. Taken together, these data suggest that the combination of two or more antigens maybe an effective vaccine development strategy to improve protection and immunogenicity against human lymphatic filariasis.

Expression of helminth genes in Leishmania: an experimental transfection system to test immunological function

Functional analysis of genes from parasitic helminths requires, at the present time, heterologous expression. We have adapted the well-characterized system of transfection in Leishmania protozoal parasites, as a means of analysing the effect of single filarial genes on the mammalian immune system. For example, testing the function of the Brugia malayi abundant larval transcript (ALT) gene-transfected Leishmania mexicana were found to be significantly more virulent in macrophages in vitro. The course of infection in vivo is also aggravated by expression of the ALT gene. Examples are also given of transgenes which reduced in vitro growth within macrophages, as well as others which exert no effect on the protozoal parasitism. Thus, Leishmania transfection provides a tractable system to analyse helminth gene function within the context of the host immune system.

Heterologous expression of the filarial nematode alt gene products reveals their potential to inhibit immune function

BackgroundParasites exploit sophisticated strategies to evade host immunity that require both adaptation of existing genes and evolution of new gene families. We have addressed this question by testing the immunological function of novel genes from helminth parasites, in which conventional transgenesis is not yet possible. We investigated two such novel genes from Brugia malayi termed abundant larval transcript (alt), expression of which reaches ~5% of total transcript at the time parasites enter the human host.ResultsTo test the hypothesis that ALT proteins modulate host immunity, we adopted an alternative transfection strategy to express these products in the protozoan parasite Leishmania mexicana. We then followed the course of infection in vitro in macrophages and in vivo in mice. Expression of ALT proteins, but not a truncated mutant, conferred greater infectivity of macrophages in vitro, reaching 3-fold higher parasite densities. alt-transfected parasites also caused accelerated disease in vivo, and fewer mice were able to clear infection of organisms expressing ALT. alt-transfected parasites were more resistant to IFN-γ-induced killing by macrophages. Expression profiling of macrophages infected with transgenic L. mexicana revealed consistently higher levels of GATA-3 and SOCS-1 transcripts, both associated with the Th2-type response observed in in vivo filarial infection.ConclusionLeishmania transfection is a tractable and informative approach to determining immunological functions of single genes from heterologous organisms. In the case of the filarial ALT proteins, our data suggest that they may participate in the Th2 bias observed in the response to parasite infection by modulating cytokine-induced signalling within immune system cells.

The Larval Specific Lymphatic Filarial ALT‐2: Induction of Protection Using Protein or DNA Vaccination

Genes from the infective stage of lymphatic filarial parasites expressed at the time of host invasion have been identified as potential vaccine candidates. By screening an L3 cDNA library with sera from uninfected longstanding residents of an area endemic for onchocerciasis, so-called "endemic normals" (EN), we have cloned and characterized one such gene termed the abundant larval transcript two (ALT-2). The stage specificity of ALT-2 gene transcription and protein synthesis was confirmed by PCR using genespecific primers, and by western blot analysis of protein extracts from various stages of the parasite life cycle using specific antisera. Significant differences in antibody response to the recombinant ALT-2 were observed in endemic populations with differing clinical manifestations of lymphatic filariasis with an antibody response present in sera from 18 of 25 (72%) EN subjects compared to 9 of 25 (36%) with subclinical microfilaracmia (MF) and 14 of 25 (52%) of those with chronic lymphatic obstruction (CP) (P=0.01 for comparison of EN to CP or to MF). This differential responsiveness suggests that the protective immunity postulated to account for their uninfected status might be associated with a response to this protein. When the utility of ALT-2 as a vaccine candidate was tested in a murine model using either recombinant protein or a DNA vaccine construct, statistically significant protection was observed when compared to a control filarial gene product expressed across all stages of the parasite lifecycle (SXP-1; P=0.02 for protein and P=0.01 for the DNA vaccine) or compared to adjuvant alone. This level of protection indicates that this vaccine is a promising candidate for further development.

Abundant larval transcript-1 and -2 genes from Brugia malayi: diversity of genomic environments but conservation of 5' promoter sequences functional in Caenorhabditis elegans.

The genomic organisation of two abundant larval transcript (alt) genes from the filarial nematode Brugia malayi has been defined. The products of these genes are 78% identical in amino acid sequence, and are highly expressed in a stage-specific manner by mosquito-borne infective larvae. alt-1 is present as two near-identical copies organised in an inverted repeat of approximately 7.6 kb, occupying a total of 16 kb of the genome. alt-2 is a single-copy gene at a different locus to alt-1. The two alt-1 genes (alt-1.1 and -1.2) are 99.7% identical in coding sequence and 99.5% in intronic sequences. Both alt-1 and -2 contain 3 introns, and the third intron of alt-2 exhibits a size polymorphism evident in different individual parasites from the laboratory-maintained strain. Genomic sequence up- and down-stream from alt-1.1/1.2 (26 and 6 kb, respectively) and alt-2 (6 and 4 kb, respectively) show that neither gene is in a multiple array or an operon. Most notably, the neighbouring genes of alt-1 and -2 show no similarity to each other, or to the genes flanking the distant alt homologue in Caenorhabditis elegans. Despite this diversity in flanking genes, the 5' UTR tracts extending some 800 bp upstream of each B. malayi alt gene show a high degree of similarity (overall 59% identity with tracts of 77-86% identity). Surmising that this region may contain conserved promoter elements, constructs containing the B. malayi alt 5' UTR with or without coding sequence were made fused to beta-galactosidase reporter protein. These constructs were injected into the syncytical gonad of C. elegans and progeny stained for beta-gal expression. Our results show relatively strong expression in the gut cells of C. elegans for both alt-1 and -2 constructs, commencing in larval worms and continuing into adulthood. Moreover, expression was enhanced when constructs contained segments of alt-1 coding and intronic sequence in addition to the 5' UTR. We conclude that the high level of alt transcription in filarial L3s is not due to expression from a multi-copy gene family but to a set of strong promoter elements shared between the two alt genes.