The therapeutic use of antisera started at the end of the 19th century, following the pioneering work of von Behring, Kitasato, Roux and Calmette. The fi rst antivenoms were developed by Calmette and Phisalix and Bertrand in 1894 (21). At the dawn of immunology, it was observed that animals immunized with specifi c toxins or venoms developed an antibody response that could be benefi cial to the treatment of many different diseases, from tetanus and diphtheria to snake bite envenomings and rabies. Thereafter, passive immunization, or serotherapy, became a powerful therapeutic tool based initially on the use of non purifi ed serum that caused a high incidence of adverse effects. http://bahankuliahkesehatan.blogspot.com/
During the 20th century, with the development of methods to purify serum proteins, fractionation protocols were introduced in the production of antisera and therapeutic preparations were obtained of either intact antibodies (IgG) or antibody fragments [F(ab')2] against antigens of clinical relevance.
High quality preparations of heterologous immunoglobulins (intact IgG molecules), or products of their enzyme digestion, can now be manufactured following methods largely in the public domain. Many laboratories prepare horse-derived antisera using various modifi ca-tions of the original Pope method (22), based on pepsin digestion and ammonium sulphate precipitation, to yield F(ab')2 antibody fragments (23, 24). Other preparations consist of intact IgG molecules, purifi ed by caprylic acid precipitation of non-IgG serum proteins (25, 26). Additional steps such as ion-exchange chromatography have been incorporated by some manufacturers (23, 24).
Manufacturers therefore use very different methods, some of which are based on tra-ditional plasma fractionation protocols, others on more complex steps, as shown at a WHO workshop convened to discuss antivenom production and control procedures (27). A recent workshop of Latin American public laboratories also highlighted the variety of techniquesused in antivenom manufacture (28) and their respective impact on product yield, safety and quality. The information available showed ample opportunities for technology transfer as well as the need for improved production processes and training of manufacturers in the developing world.
A list of antivenoms available in 1995 was prepared by Meier (29) but several labora-tories have stopped manufacture since then. Currently, many laboratories face signifi cant diffi culties in pursuing the manufacture of antisera, improving the quality of the products or increasing the production capacity. Some production centres require extensive upgrading of the infrastructure, equipment and manufacturing technologies, to meet required quality and safety standards, others lack qualifi ed staff. Those with more advanced manufacturing methods require a clearer defi nition of the size and markets needs in order to design reliable long-term manufacturing strategies.
The diverse scenarios outlined above support the development of a worldwide strategy to increase antiserum production to respond to clinical needs. A detailed analysis of the situation is required together with the design and implementation of different approaches which should be adapted to product needs.
STRENGTHS AND WEAKNESSES OF CURRENT
ANTISERA MANUFACTURE
Technologies for the fractionation of animal serum and the purifi cation of intact IgG or F(ab')2 fragments are available in the public domain and production protocols have been reported in international publications. Relevant guidelines on the principles of GMP are also available (30) and can be adapted to the manufacture of animal-derived antisera. The openness of this technological fi eld, and the fact that many groups involved in research, development and production of antisera are public institutions, brings further possibilities for the establishment of a dynamic process of training and transfer of technology . In the case of envenomings by snake bites and scorpion stings, there is extensive scientifi c knowledge on the clinical, pathophysiological, biochemical and immunological characteristics of venoms. The species responsible for most snake and scorpion envenomings in the different regions of the world have been identifi ed and many of their venoms partially characterized. There is also abundant scientifi c literature on the cross-reactivity of antivenoms against ven-oms from different species of snakes within a specifi c geographical region. Such information can be directly applied to the design of immunizing mixtures to raise effective neutralizing antisera against the most relevant venoms from a given area or geographical region.
With regard to the rabies virus, this is an excellent immunogen which readily induces strong immune responses in horses, facilitating the preparation of rabies immunoglobulins. In addition, the fi eld of human immunoglobulin preparations for intravenous use has witnessed great advances in the plasma fractionation methodologies and viral reduction procedures introduced in the production processes (31, 32). This knowledge can be helpful to the manufacturers of animal-derived antisera in order to improve the quality and safety of these products.
On the other hand, the production of antisera faces diffi culties that need to be addressed and solved to guarantee adequate global supply. The most important weaknesses are:
(1) low volumes of production;
(2) poor safety and effi cacy of some products and;
(3) defi cient or non-existent regulation and control of antisera in some countries
Many manufacturers in the public sector operate on a small production scale, and, as such, are unable to satisfy the national demand. This highlights the need for substantial investment in equipment, infrastructure and training of technical and administrative staff to ensure self-suffi ciency.
ANTISERUM POTENCY
Effective treatment of rabies and envenoming is critically dependent on the availability of good quality antisera. Defi cient quality assurance and quality control practices, together with the lack of regulatory policies in some countries, result in the production or impor tation of antisera with low neutralizing potency. Ineffective antivenoms may also be prepared because of an inappropriate selection of the venoms used as immunizing mixtures. This illustrates a lack of information on the snake fauna of the area or region as well as on the composition and immunochemistry of venoms. The problem is aggravated by the lack of preclinical control of many antivenoms.
The neutralization by antivenoms of the most relevant toxic activities of the venoms with greatest medical signifi cance in a particular territory should be assessed. For example, several groups in Latin America have succeeded in the preclinical characterization of antivenoms against venoms from different areas in the region. As a result, the neutralizing ranges of many antivenoms have been established and used to support the distribution of antivenoms within, and occasionally, among countries (33, 34). However, this information is lacking for many antivenoms and venoms throughout the world.
The control of the biological activity of antivenoms depends, among others, on the preparation of representative venom pools obtained from the snakes and scorpions species targeted (27). This requires concerted efforts among zoologists, toxinologists, manufactur-ers and regulators to establish protocols for the maintenance in captivity (and ideally for the reproduction) of snakes and scorpions, the appropriate venom collection and storage, the design of representative venom pools and the testing of venoms toxicity. In order to guarantee an appropriate geographical spectrum of effi cacy of an antivenom, it is essential to know in which parts of the country or region the specifi c envenoming is predominant. The control of the neutralizing ability of an antivenom preparation should be performed using pools of well-characterized venoms, taking into account the known causes of intra-specifi c variation in venom composition and antigenicity (35). Currently, there are many lacunae in the preparation and use of venom pools for antivenom standardization and control. This explains the discrepancies in the potency tests carried out in different laboratories. To solve this problem, a network of quality control laboratories should be formed and the sharing of standard venoms for use in assays should be encouraged (36,37). The experience in Brazil, where a well-defi ned national standard venom of the jararaca snake Bothrops jararaca is prepared and distributed to manufacturers and quality control laboratories, is a good example of national and inter-laboratory coordination.
The regulatory overview and quality control of rabies immunoglobulin is poor or absent in some countries. Preclinical characterization can provide only preliminary guidance about therapeutic effi cacy which can be established only through clinical studies or post-marketing surveillance information.
ANTISERUM SAFETY
Antiserum safety is another aspect that demands careful at tention. Upon parenteral administration, antisera may induce early or late adverse reactions.
Early adverse reactions (EARs):
Intravenous administration of antisera results in EARs in a variable proportion of patients. These are best categorized as anaphylactic reactions. Clinical features include urticaria, itching, fever, tachycardia, vomiting, abdominal colic, headache, bronchospasm, hypoten-sion and angioedema (38, 6). The incidence of EARs depends on the quality, dose, protein content, route of administration and speed of intravenous injection or infusion (38). Unless patients are observed closely during at least 2 hours after intravenous antivenom administration, EARs may not be detected. This lack of surveillance leads to underreporting of side effects. With antivenoms of good quality profi le, there is a low incidence (less than 10%) of generally mild EARs, mostly urticaria and itching. However, for other products, the incidence of such reactions may be as high as 85%, including potentially life-threatening systemic disturbances such as hypotension and bronchospasm (6). EARs are attributable, in part, to the physicochemical characteristics of the particular antivenom preparation. The presence of protein aggregates is believed to contribute to complement activation (39) and to the onset of EARs (38). The formation of such aggregates often refl ects defi ciencies during fractionation or freeze-drying of products. Likewise, the presence of contaminant proteins contributes to the reactogenicity of antisera, as well as the total amount of protein administered which is related with the incidence of both EARs and late antivenom reac-tions (LAR). The incidence of EARs would not be attributable to the use of intact IgG, since antivenom immunoglobulin preparations purifi ed by caprylic acid fractionation of horse
plasma present a good safety profi le (40). Equine rabies immunoglobulin has proven extremely safe with a reaction rate of 1.13% (41) because it is never administered intravenously and the total amount of equine protein injected is relatively low. Some antisera carry the risk of causing pyrogenic reactions, implying poor manufacturing practices (6).
Late adverse reactions (LARs):
These resemble classical serum sickness and are also described as a consequence of antiserum therapy. Their true incidence is poorly known, mostly because patients leave health centres within the fi rst few days after treatment, and the manifestations of serum sickness do not appear until 7-14 days post-treatment. However, in one series of patients who received a poorly refi ned antivenom and where a thorough follow-up was possible, it was shown that the incidence of serum sickness increased to almost 100%, proportionally to the total dose of antivenom infused and, the interval between treatment and the appearance of symptoms decreased (42).
There have been no reports of infectious diseases transmitted to humans by the admin-istration of animal antisera, but the microbiological safety of these products is of growing concern. There is an urgent need to validate the capacity for viral removal and/or inactivation that can be achieved by currently-used manufacturing processes of antisera. Preliminary results from a limited number of studies suggest that some of the production steps currently used, such as acid pH, pepsin digestion, caprylic acid precipitation and possibly others, canbe effective in virus reduction (43, 32). However, this area requires signifi cant collaborativeefforts among manufacturing laboratories and research groups, to perform viral validation studies and transfer of know-how for correct implementation.
Problems associated with poor safety of some antisera preparations are clearly linked tofailures or lack of GMP. The principles of GMP should cover all steps in antiserum production,including the handling and care of animals used for immunization, the preparation of the appropriate venom and immunization protocols, the bleeding of horses, the blood and plasma collection procedures, the plasma fractionation process as well as the steps of aseptic fi llingand freezedrying of the fi nal product. Similarly, the production of water, the cleaning and sanitisation of equipment and clean rooms, and the design of all production systems shouldstrictly follow GMP principles. Failure to ful fi l these requirements results in poor qualityand safety profi les. These problems are also associated with defective training of the staffinvolved in antiserum production, lack of technological innovation and lack of investment inthe implementation of GMP.
There are ample opportunities for improving the production of antisera at a global level.A WHO coordinated training programme should be established for strengthening techni-cal expertise in local and regional laboratories aimed at the implementation of GMP in allmanufacturing facilities.
CLINICAL STUDIES
The serious lack of well-designed, controlled clinical trials in the evaluation of the effi cacy and safety of antivenoms is a defi ciency that needs to be addressed at a global level.Some clinical studies have been published, the fi rst in 1974 (44), and provided valuable information on relative effi cacy of various antivenoms, initial dosage, pharmacokinetics and safety profi le (40,45). Quite often, antivenoms are introduced in a given country or region without appropriate clinical validation of their effi cacy or safety and in absence of
regulatory oversight. This may have serious public health implications. The reported use of counterfeit products and imported geographically-inappropriate non-specifi c antivenoms in some countries, notably in Africa, further aggravates the problem. The therapeutic failure of these products leads to a loss in confi dence in medical treatment of envenomings within the population and a return to the use of ineffective and sometimes dangeroustraditional methods.
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