Pokharel DR et al. (2006) Vaccine "Vaccination with Setaria cervi 175 kDa collagenase induces high level of protection against Brugia malayi infection in jirds."

Pokharel DR, Reddy MV, Rathaur S, Rai R, Nandakumar Kodumudi K

[Vaccine, 2006]

A zinc containing metalloprotease, 175 kDa collagenase, purified from adult female Setaria cervi showed strong cross-reactivity with sera from putatively immune (PI) individuals (unpublished observation) and induced cytotoxicity to B. malayi L3 larvae and microfilariae by ADCC mechanism [Srivastava Y, Bhandari YP, Reddy MVR, Harinath BC, Rathaur S. An adult 175 kDa collagenase antigen of Setaria cervi in immunoprophylaxis against Brugia malayi. J Helminth 2004;78:347-52]. These preliminary observations suggested the immunoprotective nature of collagenase. To confirm the vaccine potential of this protease, a vaccine trial was conducted in jirds (Meriones unguiculatus) against human filarial parasite B. malayi. The vaccination resulted into a mean protection level of 75.86% and produced high level of protease neutralizing antibodies. Cytokine analysis in immune jirds sera suggested a mixed Th1/Th2 type cellular immune response whereas ELISA, immunoblotting and enzyme antibody inhibition assay revealed the presence of specific anti-collagenase antibodies. Taken together, all these results suggest that S. cervi 175 kDa collagenase could form the basis of an effective molecular vaccine against human lymphatic filariasis.

Cheirmaraj K et al. (1990) J Immunoassay "Diagnostic use of polyclonal antibodies raised in mouse ascitic fluid in bancroftian filariasis."

Harinath BC, Reddy MV, Cheirmaraj K

[J Immunoassay, 1990]

Polyclonal antibodies were produced against Brugia malayi adult antigens (BmA (PBS) SAg and BmA (SDS) SAg) in mouse ascitic fluid by immunising Balb/c mice intraperitoneally with high ratio of adjuvant to immunogen. The diagnostic use of these antibodies in detecting circulating filarial antigen in bancroftian filariasis was studied by sandwich enzyme-linked immunosorbent assay (sandwich ELISA) using stick assay system. Both antibodies raised against PBS and SDS soluble antigens were found to be equally sensitive and relatively specific in detection of circulating filarial antigen. When anti BmA (PBS) SAg antibody was used in sandwich ELISA, 90% of microfilaraemic sera, 30-40% of acute and sub acute filarial sera, 20% of chronic filarial sera, 7% of endemic normal sera and none of 15 non-endemic normal sera were positive for filarial antigen. Using anti BmA (SDS) Sag antibody, 93% of microfilarial sera, 40% of acute and sub acute filarial sera, 20% of chronic filarial sera and none of 15 endemic and non-endemic normal sera showed the presence of filarial antigen. The filarial antigen detection using anti BmA S Ag antibodies produced in mouse ascitic fluid in sandwich ELISA may be useful in detection of active stage (microfilaraemia) of infection.

Vasu C et al. (2000) Parasitol Int "A 43-kDa circulating filarial antigen fraction of Wuchereria bancrofti in immunoprophylaxis against Brugia malayi in jirds."

Reddy MV, Vasu C, Harinath BC

[Parasitol Int, 2000]

A 43 kiloDaltan (kDa) antigen fraction (CFA2-6) isolated from microfilaraemic plasma of bancroftian filarial patients showed selective reactivity with sera samples collected from endemic normals. Antibodies raised against this antigen showed a strong reactivity with the surface of Brugia malayi infective larvae as well as microfilariae. Similar antigenic determinants were detected in the parasite extracts, but not in the excretory-secretory products. Further analysis was done on the immunoprophylactic potential of CFA2-6 in inducing immunity against Brugia malayi in Meriones unguiculatus (jird) in vivo. A strong protective response of approximately 84% was observed against the development of the filarial parasite in the jirds immunized with CFA2-6. The immunized jirds also showed a significant clearance (87%) of microfilariae inoculated intravenously. Approximately 65% of infective larvae failed to survive in jirds transferred with anti-CFA2-6 serum compared to the jirds transferred with sera from the control jirds. Passive transfer of anti-CFA2-6 antibody to the jirds followed by intravenous inoculation of microfilariae resulted in the reduction of 77% of circulating microfilariae. This study suggests that the 43-kDa CFA2-6 could stimulate a strong protective immune response against infective larvae and microfilariae in experimental animals.

Dabir S et al. (2008) Vaccine "Prophylactic evaluation of recombinant extracellular superoxide dismutase of Brugia malayi in jird model."

Goswami K, Reddy MV, Dabir P, Dabir S

[Vaccine, 2008]

The immunoscreening of Brugia malayi adult cDNA library with pooled endemic normal sera identified several seroreactive clones including, EC-SOD which contained a 612 bp insert and showed significant nucleotide and deduced amino acid sequence homologies with superoxide dismutase (SOD) of other nematode parasites. The SODs are known to play an important role in the protection of parasite against reactive oxygen species of the host. The coding region of the B. malayi EC-SOD (BmEC-SOD) was cloned and expressed in Escherichia coli followed by affinity purification on nickel agarose resin. Staining of native polyacrylamide gel for SOD activity of the expressed recombinant protein revealed that SOD activity inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide, indicating presence of Cu/Zn-SOD. The rBm EC-SOD protein showed its activity over a broad range of pH.7.0-11.0. Further the immune protective activity of recombinant EC-SOD antigen was evaluated in susceptible host, jirds (gerbils) (Meriones unguiculatus) against B. malayi filarial infection. The immunized jirds showed 33.5% and 36% cytotoxicity against microfilariae and 42.8% and 45.5% cytotoxicity against infective larvae in in vitro antibody dependent cellular cytotoxicity (ADCC) assay and in in situ micropore chamber methods respectively. This study suggests that the rBm EC-SOD antigen could stimulate a partial protective immune response against microfilariae and infective larvae in experimental animals against filarial infection.

Anand SB et al. (2011) J Helminthol "A combination of two Brugia malayi filarial vaccine candidate antigens (BmALT-2 and BmVAH) enhances immune responses and protection in jirds."

Kaliraj P, Reddy MV, Anand SB, Kodumudi KN

[J Helminthol, 2011]

In this study filarial recombinant protein or DNA vaccine constructs encoding BmALT-2 and BmVAH as single or as cocktail antigens were evaluated. Male jirds were immunized intramuscularly with DNA vaccine constructs or were immunized intraperitoneally with protein vaccine. The single and bicistronic DNA constructs induced substantial interferon- responses in spleen cells; antigen-specific responses were higher following immunization with the bicistronic cocktail construct and evoked a significant protective response of 57% in jirds challenged with Brugia malayi that was similar in the antibody-dependent cellular cytotoxicity (ADCC) assay and micropore chamber experiment. The cocktail protein vaccines induced a mixture of IgG2a (Th1) and IgG1 (Th2) responses with 80% protective response when challenged with B. malayi infective larvae. However, the single protein vaccine rALT-2 induced Th2 (IgG1/IgG3) with a 70% protective response and rVAH induced Th1 (IgG2a) with a lower proliferative response with 60% protection following challenge with B. malayi infective larvae. These results suggest that filarial cocktail protein vaccines are able to elicit substantial immune and protective responses when compared with single antigen vaccination in suitably vaccinated jirds.

Dakshinamoorthy G et al. (2013) Vaccine "Multivalent fusion protein vaccine for lymphatic filariasis."

Dakshinamoorthy G, Kalyanasundaram R, Reddy MV, Samykutty AK, Munirathinam G

[Vaccine, 2013]

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.

Gupta S et al. (2005) Exp Parasitol "Setaria cervi: immunoprophylactic potential of glutathione-S-transferase against filarial parasite Brugia malayi."

Harinath BC, Bhandari YP, Rathaur S, Reddy MV, Gupta S

[Exp Parasitol, 2005]

Glutathione-S-transferase (GST) has been detected in the adult female Setaria cervi, a bovine filarial parasite. The role of S. cervi GST antigen in inducing immunity in the host against Brugia malayi microfilariae and infective larvae was studied by in vitro antibody dependent cell mediated reaction as well as in situ inoculation of filarial parasites within a microchamber in Mastomys. The immune sera from glutathione-S-transferase immunized Mastomys promoted the adherence of peritoneal exudate cells to B. malayi microfilariae and infective larvae in vitro inducing 80.7 and 77.6% cytotoxicity, respectively in 72 h. In the microchambers implanted in the immunized Mastomys host cells could migrate and adhere to the microfilariae and infective larvae and induced 77.8 and 75% cytotoxicity to B. malayi microfilariae and infective larvae in 72 h, respectively. These results suggest that native GST from S. cervi is effective in inducing protection against heterologous B. malayi filarial parasite and thus has potential in immunoprophylaxis.

Krithika KN et al. (2005) Indian J Exp Biol "Identification of 38kDa Brugia malayi microfilarial protease as a vaccine candidate for lymphatic filariasis."

Anandharaman V, Krithika KN, Reddy MV, Kulkarni S, Dabir P

[Indian J Exp Biol, 2005]

A FPLC purified 38kDa protease (Bm mf S-7) isolated from B. malayi microfilarial soluble antigen was identified. It showed pronounced reactivity with sera collected from 'putatively immune' asymptomatic and amicrofilaraemic individuals residing in an endemic area for bancroftian filariasis. Further the immune protective activity of Bm mf S-7 antigen was evaluated in susceptible hosts, jirds (Meriones unguiculatus) against B. malayi filarial infection. The antigen showed 89% cytotoxicity against mf and 87-89% against infective (L3) larvae in in vitro antibody dependent cellular cytotoxicity Assay (ADCC) and in situ micropore chamber methods. Bm mf S-7 immunized jirds after challenge infection showed 81.5% reduction in the adult worm burden. The present study has shown that, the 38kDa microfilarial proteases (Bm mf S-7) could stimulate a strong protective immune response against microfilariae and infective larvae in jird model to block the transmission of filariasis. Analysis of IgG subclasses against Bm mf S-7 revealed a significant increase in IgG2 and IgG3 antibodies in endemic normals. Lymphocyte proliferation to Bm mf S-7 was significantly high in endemic normal group as compared to that in clinical and microfilarial carriers. Significantly enhanced levels of IFN-gamma in the culture supernatant of PBMC of endemic normals followed by stimulation with Bm mf S-7 suggest that the cellular response in this group is skewed towards Th 1 type.

Srivastava Y et al. (2004) J Helminthol "Adult 175 kDa collagenase antigen of Setaria cervi in immunoprophylaxis against Brugia malayi in jirds."

Srivastava Y, Harinath BC, Rathaur S, Bhandari YP, Reddy MV

[J Helminthol, 2004]

A 175 kDa antigen fraction with collagenase activity was isolated and purified from somatic extracts of adult Setaria cervi females using column chromatography involving consecutive steps of DEAE-Sepharose CL6B and Sephadex G-100. The optimum pH for 175 kDa collagenase was found to be pH 7.0. Sensitivities to a variety of inhibitors and activators indicated that the 175 kDa coIlagenolytic enzyme was metalloserine in nature. The enzyme hydrolysed a variety of protein substrates such as haemoglobin, casein, azocasein (general substrates) and collagen, FALGPA (furanoyl-acryloyl-leu-gly-pro-ala), the specific substrate of collagenase. The enzyme showed 57% inhibition by jird anti-somatic collagenase antibodies and reacted insignificantly with normal jird sera. Further analysis was undertaken on the immunoprophylactic potential of 175 kDa collagenase in inducing immunity against Brugia malayi (a human filarial parasite) in jirds (Meriones unguiculatus) in vitro and in situ. Immune sera of jirds raised against this antigen promoted partial adherence of peritoneal exudate cells to B. malayi microfilariae (mf) and infective larvae (L3) in vitro and induced partial cytotoxicity to the parasites within 48 h. The anti-S. cervi 175 kDa antigen serum was more effective in inducing cytotoxicity to B. malayi L3, than mf. In the microchambers implanted inside immune jirds, host cells could migrate and adhere to the mf and infective larvae thereby killing them partially within 48 h.

Jospin M et al. (2002) J Physiol "Characterization of K(+) currents using an in situ patch clamp technique in body wall muscle cells from Caenorhabditis elegans."

Jospin M, Allard B, Mariol MC, Segalat L, Segelat L

[J Physiol, 2002]

The properties of K+ channels in body wall muscle cells acutely dissected from the nematode Caenorhabditis elegans were investigated at the macroscopic and unitary level using an in situ patch clamp technique. In the whole-cell configuration, depolarizations to potentials positive to -40 mV gave rise to outward currents resulting from the activation of two kinetically distinct voltage-dependent K+ currents: a fast activating and inactivating 4-aminopyridine-sensitive component and a slowly activating and maintained tetraethylammonium-sensitive component. In cell-attached patches, voltage-dependent K+ channels, with unitary conductances of 34 and 80 pS in the presence of 5 and 140 mm external K+, respectively, activated at membrane potentials positive to -40 mV. Excision revealed that these channels corresponded to Ca2+-activated K+ channels exhibiting an unusual sensitivity to internal Cl- and whose activity progressively decreased in inside-out conditions. After complete run-down of these channels, one third of inside-out patches displayed activity of another Ca2+-activated K+ channel of smaller unitary conductance (6 pS at 0 mV in the presence of 5 mm external K+). In providing a detailed description of native K+ currents in body wall muscle cells of C. elegans, this work lays the basis for further comparisons with mutants to assess the function of K+ channels in this model organism that is highly amenable to molecular and classical genetics.