Introduction
Envenomation from snakebite is a significant public health issue. Snake venom injection can be related to simultaneous processes, including the toxic effects of the venom toxins and the stimulation of the immune response to produce an antibody towards the venom component. The secret to producing effective antivenoms for animals is to stimulate the immune system to identify and neutralize the deadly proteins found in venom [1]. Snake venoms contain mixtures of more than 100 proteins with different molecular weights and biological activities [2]. Elapid bites, including those from kraits and cobras, are thought to be neurotoxic because they result in neuromuscular paralysis that is mediated by low molecular weight toxins (6–9 kDa) belonging to the three-finger toxins (3FTs). The most important toxins among the 3FTs are the postsynaptic neurotoxins. Postsynaptic neurotoxins, which attach to nicotinic acetylcholine receptors at the neuromuscular junction, are the most significant toxins among the 3FTs [3].
Toxin binding results in respiratory failure, muscle paralysis, and inhibition of neuromuscular transmission. Another important class of 3FTs is the cytotoxins, also referred to as cardiotoxins. They mostly have cytolytic action and induce local tissue necrosis. The molecular size of venom antigens can determine their antigenic properties. The high molecular weights, mostly hydrolytic enzymes, are usually not lethal. Major snake toxin components, including neurotoxins, phospholipase A2 (PLA2), metalloproteinase (MP), and elapid snake disintegrins, are low molecular size (5–30 kDa) components. However, the low molecular size of these proteins represents a problem with generating antibodies, but fewer immunogens, owing to their low immunogenicity [4].
One of the most significant venomous snakes that contributes to snakebite incidents in Southeast Asia, particularly Thailand, is the cobra (genus Naja). Production of therapeutic antivenoms against the elapids, especially cobra (Naja kaouthia), has proven very difficult because of low immunogenicity related to low molecular size (7.8 kDa) [5]. Developing complementary strategies to improve the antibody response could be an alternative and useful approach to creating better therapeutic antivenoms with higher neutralizing potencies. Furthermore, the generation of an effective immune response to recognize and neutralize toxic proteins of venoms is the key component in the production of effective antivenoms in animals [6].
The goal of this study is to boost the humoral immune response to modified cobra venom by developing four straightforward immunization strategies using crude cobra venom and toxin fraction (<10 kDa). It is important to note that the preparation of the antivenom will target the neutralization of fatal neurotoxins (<10 kDa) rather than high molecular weight enzymes. A rabbit model will be used to assess particular cell immunology and cytokine mediators as biomarkers for pertinent immune response [7]. The creation of more effective antivenoms may be aided by important knowledge regarding the impact of synergistic methods between snake venom and toxin fraction [8,9].
Materials and Methods
Snake venom and toxin fraction
The Snake Farm, Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok, provided the lyophilized cobra venom. To prepare the toxin fraction, the venom was dissolved in sterile normal saline, resulting in a final protein content of 2.0 mg/ml. The venom solution was filtered through a 10 kDa molecular weight cut-off (MWCO) ultra-filtration membrane at 9000 rpm for 10 minutes at 4°C to remove high molecular weight venom proteins. Toxin fraction was the name given to the filtrate. Using the mass spectrometry method, the protein compositions of the filtrate and retentate were identified, and their volumes were noted. The peptides were analyzed by MS/MS using micrOTOF-Q IITM ESI-Qq-TOF mass spectrometer (Bruker; Berman, Germany) equipped with an online nanoESI source. Protein identification was performed using the MASCOT search engine (http://www.matrixscience.com) and queried against the SWISSPROT database.
Animals
Male ICR mice weighing 18-20 g were supplied by the Laboratory Animal Center, Mahidol University. Mice were housed at 25°C and had free access to food and water. Male New Zealand white rabbits weighing 2.5 kg were housed individually with free access to food and water. All animals used in this study were maintained and treated under strict ethical conditions by the “International Animal Welfare Recommendations” and approved by the Queen Saovabha Memorial Institute Animal Care and Use Committee (QSMI-ACUC-02-2020).
Immunization strategies
According to the animal strategies approved by the ethical committee, a total of 12 New Zealand white rabbits were used. They were divided into four simple immunization strategies containing three rabbits per group; A: crude cobra venom, B: cobra toxin fraction (< 10 kDa), C: crude cobra venom following with its < 10 kDa fraction, and D: the mixture 1:1 of crude cobra venom and its < 10 kDa fraction. The rabbits were given five booster doses at two-week intervals. The crude venom/toxin fraction was then combined with Freund's complete adjuvant (or incomplete Freund's adjuvant) at a 1:1 (v/v) ratio in the first and second injections. The mixture was homogenized thoroughly with a three-way connector and two glass syringes. The last three vaccines included the use of alum (aluminum hydroxide) as an adjuvant. Crude venom and toxin fraction were combined with alum at a 1:1 (v/v) ratio and stored on ice until use. The immunization was performed utilizing a low dosage, low volume, and multi-site approach. The immunogen was injected subcutaneously at five sites, with a volume of 0.2 ml at each site. Blood samples were taken from each rabbit every two weeks. Flow cytometry and a CBC blood test were used to determine the number of B and T cells in whole blood samples. Peripheral Blood Mononuclear Cells (PBMC) were isolated from whole blood in the EDTA for RNA extraction. The serum from each rabbit was obtained by centrifuging the clotted blood at 1500 rpm for 10 minutes at room temperature. The serum was stored at -20°C until it was utilized to neutralize lethality.
Lethality of cobra venom
The median lethal dose (LD50) of the cobra venom and toxin fraction was determined by intravenous (IV) injection into ICR mice (18-20 g, n = 5 per dose). The survival ratio was recorded after 24 hours, and LD50 was calculated using the Probit analysis method.
Cytokine primer design
Oligonucleotide primers for interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and internal control housekeeping genes: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Cyclophilin A (CycA) were developed based on rabbit cytokine sequences deposited in the GenBank database (Table 1). Primer sets were designed and optimized using Primer 3's default parameters. Eight primer pairs were chosen based on the following criteria: annealing temperature of 52°C, primer length of 18-24 bp, and amplicon size of 100-400 bp.
RNA extraction
RNA was isolated from rabbit PBMC using 400 µ l of TRIzol Reagent (Molecular Research Center, Inc, Cincinnati, Ohio, USA), following the manufacturer's instructions. The isolated RNA was suspended in 20 µ l of nuclease-free water and kept at -70°C for analysis. The concentration and purity of the extracted total RNA were measured using a spectrophotometer to measure the absorbance ratio at 260 nm and 280 nm.
Cytokine gene expression using quantification SYBR Green real-time RT-PCR (RT-qPCR)
For cytokine gene expression in rabbits, a quantitative SYBR Green real-time RT-PCR was performed using the Light Cycler (CFX 96 TouchTM Real-Time PCR) and the iTaq Universal SYBR Green One-Step kit (BioRad, CA, USA). Five microliters of total RNA were added to 15 µ l of the reaction mixture, which included 2x iTaq universal SYBR Green reaction mix, iScript reverse transcriptase, 10 pmol of each primer, and nuclease-free water. The reaction was carried out under the following conditions: 95°C for 3 minutes, followed by 40 cycles of 95°C for 10 seconds, 52°C for 10 seconds, and 72°C for 30 seconds. Following amplification, a melting curve examination was conducted. This is done to ensure the specificity of the PCR product by examining melting temperatures. The melt curve treatment was followed by 10 seconds at 95°C, then 5 seconds at 0.5°C increments between 65°C and 95°C. The housekeeping genes GAPDH and CycA were amplified using the identical conditions as described previously. The comparative threshold cycler (Ct) approach, commonly known as the 2-ᵟᵟ Ct method, is a popular method for presenting relative gene expression.
Amount of B and T lymphocytes analysis by flow cytometry
A microliter of anti-B cells was added to 50 µ l of whole blood with EDTA and gently mixed, then incubated at room temperature. After 20 minutes, mix 5 µ l of anti-T cell with 1 µ l of the second antibody and incubate for another 20 minutes. The red blood cells were then lysed for 10 minutes with 1 ml of FACS lysing solution. The staining and lysing procedures were performed in the dark at room temperature. The mixture was centrifuged at 1500 rpm and rinsed three times in PBS. The particular B and T lymphocytes were counted and analyzed with FACSCalibur flow cytometry and CellQuest Pro software (Becton Dickinson; Franklin Lanes, NJ, USA). In this approach, 1 ml of rabbit serum was combined with varied venom concentrations and incubated for 1 hour. The combination was then given to a mouse by intravenous (IV). After 24 hours, the percentage of mice that survived was determined by the rabbit serum's capacity to neutralize both crude venom and toxin fraction.
Statistical analysis
The data is displayed using the mean standard deviation (SD) for three animals. The groups were compared using a Student’s t-test enabled by software in Biostatistics version 3.02, PRIMER (The McGraw-Hill Companies, Inc, San Francisco, California, USA). Probabilities below 5 % (P < 0.05) were considered statistically significant. Four groups of immunized rabbits were compared using a One-Way Analysis of Variance (ANOVA), followed by a post-hoc Tukey HSD (Honestly Significant Difference) Test Calculator for comparing multiple treatments.
Results
Venom preparation and lethality
Lyophilized (freeze-dried) cobra venom was used in this study because snake venom is easily handled, degrades readily, and can be measured in dry weight. Thirteen toxins from cobra venom were filtered through a 10 kDa molecular weight cut-off (MWCO) ultrafiltration membrane at 9000 rpm for 10 minutes at 4°C. The small protein contents were detected and characterized using the mass spectrometry method. Nearly all toxins, including neurotoxins (cobrotoxins) and cytotoxins, have low molecular weight components ranging from 6.649 to 9.908 kDa (Table 2). Alpha-cobratoxin, nakoroxin, and muscarinic toxin-like proteins ranked first through third. A few acidic phospholipase A2 molecules (16.005 kDa) have also been discovered. In this investigation, cobra venom and toxic fraction had a median lethal dose (LD50) of 4.28 µg/mouse (body weight 18-20 g). The LD50 was obtained using the Probit analysis approach.
Complete Blood Count (CBC) profile
The CBC of rabbits was assessed before and after immunization with various techniques. The CBC profile of rabbits followed the same trend. Most CBC parameters change after 2 weeks of venom administration. Blood morphological abnormalities were observed in rabbits from D14 to D42, and then recovered to normal following immunization. Surprisingly, the amount of B lymphocytes was large, whereas neutrophil counts were low. Statistical analysis was performed to compare lymphocyte counts over many days and four distinct immunization strategies. No significant difference in lymphocyte count was observed between the groups in this study over several days (Tables 3A through 3D).
Specific T and B lymphocytes
B lymphocytes from all strategies were affected by increasing D14 following venom administration. The C and D strategies contained the most B lymphocytes, followed by the A and B strategies. All strategies were reduced from D28 and kept low on the bottom line until D98. However, A and B strategies resurfaced at D42 and D58, respectively (Fig 1A). In contrast, the number of T lymphocytes in all strategies followed the same pattern, with a slight increase and decrease from the beginning to the endpoints (Fig 1B).
Neutralization activity
The study found that crude cobra venom (7.6 and 6.9 µg/mouse, 18-20 g) could kill 88 % and 80 % of mice, respectively (Fig. 2A). The effectiveness of neutralizing these venoms in rabbit serum was also evaluated (Fig. 2B). Rabbit serum is highly effective in both C and A strategies. However, the D and B strategies had minimal impact.
Cytokine gene expression using quantification SYBR Green real-time RT-PCR (RT-qPCR) Quantification SYBR Green real-time RT-PCR (RT-qPCR) was used to assess pro- and anti-inflammatory cytokine gene expression in rabbits following four simple immunization strategies (Fig 3 and 4). The optimized primer sets had a concentration of 10 mM/µl. All primer sets were annealed at 52°C and cycled in parallel in the same real-time thermocycler. The melt curve protocol was 95°C for 10 seconds, followed by 5 seconds at 0.5°C increments from 65°C to 95°C. The extracted total RNA concentration was 100 ng. The data analysis was completed in 2 hours, therefore, it was less time-consuming. There are statistically significant differences, P < 0.05, between four immunization strategies and date-times ranging from 0 to 161 days (Supplement 1). In both strategies, pro-inflammatory cytokines IL-6 increased by 2 to 6 folds between D28 and D140 after the second dose was administered. However, the A method had lower gene expression than the other strategies. D28, D84, and D140 revealed a statistically significant difference between the strategies. Between D42 and D84, all strategies showed a high number of IFN expressions, although only D58 differed statistically. Except for approach D, all strategies showed a relatively low number of TNF expressions. TNF gene expression for strategy D increased from D2 after the first immunization to D140 before recovering to normal levels, with significant differences found in D98 and D140. Furthermore, the gene expression level of the pro- and anti-inflammatory cytokine IL-2 increased from D2 to D161 in nearly all. Furthermore, the gene expression level of the pro- and anti-inflammatory cytokine IL-2 increased from D2 to D161 in nearly all strategies. However, the gene expression levels of anti-inflammatory cytokines, IL-4 and IL-10, increased significantly in the D strategy from D2 to D161. Other ways increased gene expression, but the levels fluctuated. There were significant differences (P < 0.05) between all techniques used on D140 and D161.
Discussion
Every year, several million humans are envenomated by animals, particularly snakes, which inject venoms containing harmful enzymes and non-enzymatic proteins into their victims. The existence of signs and symptoms associated with venom toxicity variations that are followed by bites. However, it has been established that other factors, including the amount of venom injected, the victim's age, and size, can all contribute to clinical symptoms. Snakebite envenomation is frequently caused by cobras (Naja spp.) in Asia and is associated with significant fatality and morbidity rates. Cobra envenomation can cause systemic neurotoxicity and local tissue necrosis at the bite site. The increased tissue damage and necrosis caused by cobra bites are ascribed to cytotoxins (CTX), which account for 20-80 % of total venom protein. The toxicity of Elapidae snake venoms is caused by three types of components: neurotoxins, cardiotoxins (or cytotoxins), and PLA2. They have been identified as the primary targets for neutralizing antibodies [10]. However, due to its low immunogenicity, traditional immunization with crude venom failed to efficiently produce neutralizing antibodies in animals targeting lethal venom components [11].
Many studies [5, 8, 10] have been undertaken to increase the efficacy and availability of antivenoms for elapid snakes. Animal immunization for snake antivenom production requires consideration of many factors influencing the immune response to snake venoms. The individual health status and genetic background of the immunized animals, route of injection, venom dose, types of adjuvants, the mixture design of venom, and the immunization protocols should be considered. It has been recommended that an effective immunization protocol is a critical step that must be taken [12]. However, fewer than 20 % of horses inoculated in Thailand with cobra (Naja kaouthia) venom achieve the antibody titer required to produce effective cobra antivenom. This was considered to be related to the poor immunogenicity of the neurotoxins and PLA2, the most lethal toxins in the venom. Despite the enormous number of horses used, it was discovered that few horses reacted to this immunization program. The specific antivenom developed in Thailand was of low potency and insufficient availability, resulting in a lack of lifesaving antivenoms. Thus, the quality of antivenoms should be improved by enhancing their ability to neutralize venom toxicity while also lowering the occurrence of adverse events following injection into victims [13].
The simple immunization strategies were designed to boost the potency of antivenom against many snake species covering a geographic area of regions. Furthermore, immunization strategies have been modified based on data collected from basic immune response trials. They include the use of low dosages of venoms in multi-site immunization regimens, the substitution of refined lethal components for crude venoms in antigenic mixes, and the application of novel immunological adjuvants. Furthermore, animal selection should focus on producing not only large antibody titers but also neutralizing antibodies with high affinity and avidity for the appropriate venom toxin epitopes [8, 14, 15]. Some literature has described the potential synergisms of the toxin subunits in snake venom [16]. The synergistic effect in combination with snake toxin subunits could be greater than the individual components. These synergistic interactions between venom protein complex subunits possibly enhance the toxic potency of the snake venom. These techniques have also been observed with the venoms of scorpions and spiders in addition to snakes [9, 17]. When it comes to producing high-quality antiserum, developing B cells, studying immunization against infections, and modeling inflammation, rabbits are the most often employed animals in biomedical research [18-21].
Considering study approaches, we attempted to examine the possibility of simple immunization strategies, either alone or in combination with crude cobra venom and a low MW fraction, in a rabbit model, resulting in improved antivenom potency for the treatment of cobra bite victims. It is suggested that the venom's lethality results from the synergistic action of numerous components. Because the venom toxins are present in the immunogen mix, the antivenom should neutralize the fatal effects of the venom. To neutralize the lethality of the venom, several synergistic-acting toxins must be neutralized simultaneously. It is believed that our preparation for simple immunization strategies will be more successful than the antivenom now utilized to treat cobra bite victims.
However, the ultra-filtration technique, which uses a 10 kDa molecular weight cut-off (MWCO) ultra-filtration membrane, is the study’s drawback, though. This technique works well with the most deadly toxins that have a molecular weight of less than 10 kDa, including elapid 3FTs (6–9 kDa). Thus, the size of the ultra-filtration membrane should take into account the MW of the deadly components from other snake venom. Our results are consistent with those of other research utilizing a combination immunization protocol. For instance, horses were immunized with a combination of venoms and toxin fractions from numerous significant elapid snakes, such as cobras and kraits, to produce a pan-specific antivenom. To extract the toxin fractions, the venoms were ultra-filtered to exclude high molecular weight, non-toxic proteins. All the studied elapid venoms could be neutralized by the horse antiserum [3]. Additionally, in a rabbit model, coral antivenom was obtained using priming doses of Micrurus frontalis venom and booster doses of synthetic B-cell epitopes derived from Micrurus corallinus toxins (3FTs and PLA2). The activities of both venoms can be neutralized by the produced antibodies [6].
Our findings in this study show that blood shape and different types of WBC from CBC were altered after two weeks of venom administration, but recovered to normal after D42. Surprisingly, they were modified from mixed immunization methods rather than single doses of crude cobra venom or low MW fraction. The humoral immune response in a rabbit model appears to be the acute response to preserving homeostasis following snake venom injection. The major role of B cells is to generate antibodies for humoral defense. Bites and venom inoculation promote the production of venom protein-specific antibodies [22,23]. The neutralization efficiency of rabbit model serum was higher in the combination immunization protocol with crude cobra venom following its <10 kDa fraction, compared to other protocols. Nonetheless, the neutralization efficiency was almost 80% of 7.6 µg/mouse, or 1.8LD50. In terms of the manufacturing procedure for the treatment, it was the lowest experiment, despite being the highest overall. As a result, issues including the host, the venom delivery method, and the concentration and ratio of the venoms in the experiment will be further investigated. Meanwhile, the cell-mediated adaptive immune response to envenomation consists of T cells releasing various cytokines in reaction to snake venom components [24].
The amounts of cytokines released by snake venom were determined using quantitative gene expression in rabbits [25]. The rabbit serum increases gene expression of proinflammatory cytokines through innate pathways, triggering inflammatory reactions crucial to host defense. TNF is a key mediator in immunological and inflammatory responses. The gene expression level of TNF-α was both up and down-regulated. TNF-α gene expression was lower than IL-6 after venom injections. IL-6 is produced by a range of cell types and has both pro- and anti-inflammatory properties, as well as involvement in several immunological responses [26,27]. The findings of this investigation revealed that IL-6 levels in all protocols increased following the first venom injection and peaked after the third booster (D28). The stable peak began to decline two weeks following the last venom injection (D58). In all procedures, IFN-γ gene expression increased from D42 to D84 after the fourth immunization. IFN-γ gene expression was lower in crude cobra venom immunization compared to cobra toxin fraction alone or combined immunization regimens. These proinflammatory cytokines will proliferate and create other cytokines, resulting in an inflammatory cascade and anti-inflammatory cytokines. IL-4 and IL-10 are anti-inflammatory cytokines that primarily govern the inflammatory response and maintain homeostasis for the proper functioning of vital organs. The current work shows that combined immunization procedures, including crude cobra venom and purified <10 kDa fraction, can promote IL-4 and IL-10 production, which undoubtedly has a modulatory effect on the host inflammatory response. The highest levels of IL-4 and IL-10 were detected on D58 post-injection. There was no difference in IL-2 levels. These findings suggest that cobra venom envenomation is influenced by both pro- and antiinflammatory cytokine responses in a time-dependent way. A balanced ratio of pro- and anti-inflammatory cytokines is required for an optimal immunological response, which can result in envenoming complications [28]. The neutralization efficiency of rabbit model serum was higher in the combination immunization protocol with crude cobra venom following its <10 kDa fraction, compared to other protocols. However, B and T cell responses are often delayed, taking days to weeks to become completely activated, whereas defense against fast venom action necessitates an immediate reaction. Large antivenom-producing animals, such as horses, can provide passive immunity against life-saving snake envenomation [29-31].
Conclusions
The current study shows that simple immunization protocols that mix crude cobra venom and purified low MW fraction are more efficient for producing cobra antivenom than single immunization protocols that use only crude cobra venom or the low MW fraction. The animals were first injected with a combination of immunization, specifically crude venom, followed by purified fractions, which may be the best strategy to increase strong humoral immunity. They could generate poly-specific therapeutic antibodies, allowing for wide-scale production while also avoiding massive doses of venom injection. It appears that the venoms should be ultra-filtered to remove high molecular weight, non-toxic proteins before extracting the toxin fractions for use as immunogens. The presence of high MW venom proteins may interfere with the formation of antibodies specific to the fatal toxins found in elapid venom. The findings support the use of pure toxins as immunogens since they contain nearly all the deadly components associated with toxin-specific antibody selection. The antivenoms should have efficient protective antibodies, which could reduce the high rate of adverse effects. To obtain a successful immunization strategy and high-quality cobra antivenom, this combination immunization protocol may be used for horse immunization in future studies.
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