Multilevel Comparison of Indian Naja Venoms and Their Cross-Reactivity with Indian Polyvalent Antivenoms.

Snake envenoming is caused by many biological species, rather than a single infectious agent, each with a multiplicity of toxins in their venom. Hence, developing effective treatments is challenging, especially in biodiverse and biogeographically complex countries such as India. The present study represents the first genus-wide proteomics analysis of venom composition across Naja species (N. naja, N. oxiana, and N. kaouthia) found in mainland India. Venom proteomes were consistent between individuals from the same localities in terms of the toxin families present, but not in the relative abundance of those in the venom. There appears to be more compositional variation among N. naja from different locations than among N. kaouthia. Immunoblotting and in vitro neutralization assays indicated cross-reactivity with Indian polyvalent antivenom, in which antibodies raised against N. naja are present. However, we observed ineffective neutralization of PLA2 activities of N. naja venoms from locations distant from the source of immunizing venoms. Antivenom immunoprofiling by antivenomics revealed differential antigenicity of venoms from N. kaouthia and N. oxiana, and poor reactivity towards 3FTxs and PLA2s. Moreover, there was considerable variation between antivenoms from different manufacturers. These data indicate that improvements to antivenom manufacturing in India are highly desirable.

Immunoblotting Identification of Diagnostic Antigens of Paragonimus westermani Type 1 for the Detection of Human Pulmonary Paragonimiasis in North East India.

Human pulmonary paragonimiasis, an emerging concern in North East India, frequently masquerades as pulmonary tuberculosis due to clinical and radiological similarities, leading to diagnostic challenges. This research aimed to harness the immunoblotting technique to discern immunodiagnostic protein antigens from both adult worm and excretory-secretory (ES) extracts of the prevalent Paragonimus westermani type 1 in Arunachal Pradesh, North East India. We studied the time kinetics of immunoreactive patterns in relation to the duration of infection in rodent models. Immunoblot analyses were also conducted using sera from ELISA-positive patients confirmed with paragonimiasis, facilitating the selection of antigenic extracts with diagnostic potential. Further, ES protein antigens were subjected to 2D immunoblot analysis and immunoreactive protein spots identified using MALDI-TOF MS. The immunoreactivity patterns of ES antigens with sera of paragonimiasis-positive patients were detailed, and specific immunoreactive protein antigens were pinpointed using peptide mass fingerprinting (MALDI-TOF). This work underscores the enhanced diagnostic accuracy when combining ELISA with immunoblotting for pulmonary paragonimiasis in regions like North East India, marked by co-existing helminth infections.

Naja kaouthia venom protein, Nk-CRISP, upregulates inflammatory gene expression in human macrophages.

Cysteine-Rich Secretory Proteins (CRISP) are widespread in snake venoms and known to target ion channels. More recently, CRISPs have been shown to mediate inflammatory responses. Involvement of potential receptor in CRISP-induced inflammatory reactions, however, remains unknown. A CRISP protein named as Nk-CRISP, was isolated from the venom of Naja kaouthia. The molecular mass of the purified protein was found to be ~25 kDa and the primary sequence was determined by MALDI TOF-TOF. The involvement of this protein in proinflammatory effects were evaluated in THP-1 macrophage-like cells. Nk-CRISP treated cells induced up-regulation of several inflammatory marker genes in dose dependent manner. Toll like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex are known to play crucial role in recognition of damage/pathogen-associated molecular patterns and activation of innate immune response. Therefore, we hypothesized that snake venom CRISP could also modulate the innate immune response via TLR4-MD2 complex. In-silico molecular docking study of cobra CRISP with TLR4-MD2 receptor complex reveals CRISP engages its cysteine-rich domain (CRD) to interact with complex. Inhibition of TLR4 signalling pathway using CLI-095 confirmed the role of TLR4 in Nk-CRISP induced inflammatory responses. Collectively, these findings imply that TLR4 initiates proinflammatory signalling following recognition of cobra CRISP and alteration of TLR4 receptor might improve or control CRISP induced inflammation.

Proteomics of Naja kaouthia venom from North East India and assessment of Indian polyvalent antivenom by third generation antivenomics.

In the present study, venom composition, toxic effects, and immunological characteristics of Naja kaouthia venom from North East India has been studied. Using RP-HPLC, venom components were separated and proteins in the fractions were identified using ESI-LC MS/MS. Proteins identified belong to 9 different snake venom protein families. Three finger toxins and PLA2 were the most abundant protein families detected by mass spectrometry analysis. The other minor proteins families identified in the venom were kunitz-type serine inhibitors, waprin, L-amino acid oxidase, CRISP, vespyrn, nerve growth factor and metalloproteinase. This proteome composition correlated with the tested enzymatic and toxic activities of the venom. Western blot and third generation antivenomics analysis using Vins polyvalent antivenom revealed immunoreactivity towards Naja kaouthia venom of North East India. Concentration-dependent immunocapturing profile carried out using RP-HPLC displayed immunerecognition of majority of venom proteins of Naja kaouthia except few three-finger toxins. Presence of such non-immunodepleted toxins apparently may affect the performance of Vins polyvalent antivenom. Thus, inclusion of antibodies of most relevant non-immunorecognized toxins in antivenom might help to improve the quality of antivenom. BIOLOGICAL SIGNIFICANCE: Envenomings by genus Naja, represent a serious medical problem in Asian countries including North east India. In North East India, Naja kaouthia is most prevalent cobra species causing a large number of fatalities. To gain deeper insight into the spectrum of medically relevant toxins, we applied proteomics approach to unveil the proteome profile of Naja kaouthia venom. The proteomic analysis divulged the presence of two major protein families: three finger toxins and phospholipases A2. In general, polyvalent antivenom is administered for Naja kaouthia envenomings, however, this venom is not included in the immunization mixtures (only Indian Big Four venoms) for production of these polyvalent antivenoms. For the first time, third generation antivenomics approach was used to decipher maximal binding capacity of Indian polyvalent antivenom against Naja kaouthia venom. Although Vins polyvalent antivenom was effective in immunocapturing majority of venom components, however, large amount of antivenom was required to immunocapture the venom proteins. Moreover, the study revealed poor immunorecognition capacity of Vins antivenom towards four three finger toxin subtypes. This may have significant impact on antivenom efficacy in treating Naja kaouthia envenomings.

Comparative analysis of Naja kaouthia venom from North-East India and Bangladesh and its cross reactivity with Indian polyvalent antivenoms.

Naja kaouthia is one of the most prevalent medically important snakes of North East India and Bangladesh responsible for most of the bite cases. In this study, an attempt was made to decipher venom variation of Naja kaouthia venom from North East India and Bangladesh. Using multidimensional methods including reverse phase HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-PAGE) and two-dimensional gel electrophoresis (2D-PAGE), the quantitative differences in venom composition have been revealed. Moreover, tested in-vitro biochemical and biological activities also exhibited differences which could be due to venom variability. Furthermore, neutralization efficacy of commercially available Indian polyvalent antivenoms (Vins, Bharat Serum, Haffkine) was evaluated and the results displayed significant differences in neutralizing efficacy between the antivenoms. Immunoblotting experiments showed antivenom molecules cross reacted with high molecular mass components while poorly reacted towards low molecular mass proteins. Immuno-depletion study demonstrated that Vins polyvalent antivenom was poor in immunocapturing the venom proteins of both North East Indian and Bangladesh origin Naja kaouthia at the ratio of 1:16 (venom: antivenom).

Purification and partial characterization of an anticoagulant PLA2 from the venom of Indian Daboia russelii that induces inflammation through upregulation of proinflammatory mediators.

The present study describes the purification and partial characterization of a basic anticoagulant PLA2 enzyme named as Rv(i) PLA2 from the venom of Indian Daboia russelii. The molecular mass of the protein was found to be 13,659.65 Da, and peptide mass fingerprinting revealed that it belongs to group II PLA2 family. The peptide sequence showed similarity to uncharacterized basic PLA2 enzyme having an accession no. of P86368 reported from Sri Lankan D. russelii. Rv(i) PLA2 exhibited strong phospholipase A2 and anticoagulant activity. It also induced expression of COX-2 and TNF-α mRNA in a dose-dependent manner in phorbol 12-myristate 13-acetate differentiated THP-1 cells, which play a crucial role during inflammation. Chemical modification of His residue in Rv(i) PLA2 with p-bromophenacyl bromide abolished the enzymatic, anticoagulant, and inflammatory activities. The result indicates that the catalytic site of Rv(i) PLA2 might play a vital role in inducing inflammation at the bite site during D. russelii envenomation.