key: cord-0756144-akixvu3j
authors: Dixit, Rashmi; Herz, Jenny; Dalton, Richard; Booy, Robert
title: Benefits of using heterologous polyclonal antibodies and potential applications to new and undertreated infectious pathogens
date: 2016-02-24
journal: Vaccine
DOI: 10.1016/j.vaccine.2016.01.016
sha: c79b541523d38fd66a8ccb50ab64c6643b76af7d
doc_id: 756144
cord_uid: akixvu3j

BACKGROUND: Passive immunotherapy using polyclonal antibodies (immunoglobulins) has been used for over a century in the treatment and post-exposure prophylaxis of various infections and toxins. Heterologous polyclonal antibodies are obtained from animals hyperimmunised with a pathogen or toxin. AIMS: The aims of this review are to examine the history of animal polyclonal antibody therapy use, their development into safe and effective products and the potential application to humans for emerging and neglected infectious diseases. METHODS: A literature search of OVID Medline and OVID Embase databases was undertaken to identify articles on the safety, efficacy and ongoing development of polyclonal antibodies. The search contained database-specific MeSH and EMTREE terms in combination with pertinent text-words: polyclonal antibodies and rare/neglected diseases, antivenins, immunoglobulins, serum sickness, anaphylaxis, drug safety, post marketing surveillance, rabies, human influenza, Dengue, West Nile, Nipah, Hendra, Marburg, MERS, Hemorrhagic Fever Virus, and Crimean-Congo. No language limits were applied. The final search was completed on 20.06.2015. Of 1960 articles, title searches excluded many irrelevant articles, yielding 303 articles read in full. Of these, 179 are referenced in this study. RESULTS: Serum therapy was first used in the 1890s against diphtheria. Early preparation techniques yielded products contaminated with reactogenic animal proteins. The introduction of enzymatic digestion, and purification techniques substantially improved their safety profile. The removal of the Fc fragment of antibodies further reduces hypersensitivity reactions. Clinical studies have demonstrated the efficacy of polyclonal antibodies against various infections, toxins and venoms. Products are being developed against infections for which prophylactic and therapeutic options are currently limited, such as avian influenza, Ebola and other zoonotic viruses. CONCLUSIONS: Polyclonal antibodies have been successfully applied to rabies, envenomation and intoxication. Polyclonal production provides an exciting opportunity to revolutionise the prognosis of both longstanding neglected tropical diseases as well as emerging infectious threats to humans.

The aims of this review are to examine the history of animal polyclonal antibody therapy use, its development into a safe and effective product and its potential future application to humans for emerging viruses and neglected tropical diseases.

A literature search was undertaken by an experienced medical librarian to identify articles on both the safety of polyclonal In 1890, von Behring and Kitasato discovered that sera from rabbits immunised against diphtheria or tetanus were able to protect exposed mice [1] . By 1894, animal derived anti-diphtheria serum was used in humans during a European epidemic [2] ; concurrently, Phisalex and Bertrand demonstrated that the blood of horses immunised with Viper aspis (the European viper) had antivenin properties [3] . Sera from both humans and animals were subsequently used for management of illness caused by viruses such as measles, varicella and the pandemic Spanish influenza of 1918 [4, 5] . Anti-rabies polyclonal antibody preparations from hyper-immunised horses were developed in the early 20th century [6] . In this pre-antibiotic era, serum therapy was also applied to bacterial infections such as pneumococcal pneumonia, meningococcal meningitis and streptococcal scarlet fever [7, 8] .

The early, crude preparations of serum were often contaminated with animal proteins; as a result, serum sickness and anaphylaxis limited their safe application. Antibiotics against bacterial infections largely superseded serum therapy in the late 1930s and 1940s [7] . During the 1960s, however, there were improvements in enzymatic digestion and purification of equine immunoglobulins, enabling safer polyclonal antibody therapies to be developed for envenomation, rabies exposure and viral infections such as hepatitis A and B, varicella-zoster virus and respiratory syncytial virus (RSV) [8] .

The development of monoclonal antibodies (mAbs) in the 1970s enabled large quantities of specific antibody to be produced [8] . The humanisation of mAbs allowed their use in the treatment of chronic disease by repeated administration. However, polyclonal antibodies still proved more effective than monoclonal antibodies in the treatment of many infectious diseases. To date, most monoclonal antibody therapies are produced for either autoimmune conditions (such as systemic lupus erythematosus or Crohns disease) or neoplastic conditions. The only licenced monoclonal antibody to an infectious target is palivizumab, for RSV [9] . In contrast to monoclonal antibodies, polyclonal therapy, by targeting multiple epitopes, can protect against epitope mutation [4] . Additionally, more may be needed of a low avidity monoclonal antibody preparation, compared with a polyclonal preparation, to neutralise a given amount of toxin or pathogen, as is generally the case for antitoxins to snake envenomation [10] .

Animal derived polyclonal antibody therapy has been successfully and safely applied to (i) medication overdoses such as colchicine and digoxin, (ii) poisoning by snake, arachnid, marine and plant toxins, and (iii) post-exposure prophylaxis against the rabies virus [11] [12] [13] [14] [15] . Polyclonal antibody products can be made in large quantities and cost-effectively to respond to the great endemic demands in Asia and Africa as well as potential pandemic situations, globally.

Adverse reactions in humans to animal-derived polyclonal antibodies are usually due to the presence of highly immunogenic animal proteins. Type 1 hypersensitivity reactions, including anaphylaxis, begin within minutes. Type 3 hypersensitivity, or serum-sickness, results from deposition of immune complexes on small vessels of the skin, joints and kidneys; this can develop at any point over the three weeks it takes to clear the injected antibodies [3] . Antibodies consist of the antigen binding (Fab) fractions, linked by disulphide bonds as F(ab ) 2 , and the crystallisable fraction (Fc). The reactogenic Fc fragment can induce complementmediated urticaria, angioedema, lymphadenopathy, arthralgia, and nephropathy [3] . Fc removal with retention of the immunogenic F(ab)/F(ab ) 2 components was first applied to digoxin antiserum in 1976 [16] . The F(ab)/F(ab ) 2 fragments were isolated initially through salt precipitation and subsequently, since the mid-1990s, via chromatographic purification [17] . Complement mediated anyphylactoid reactions, fever and hypersensitivity have been reduced by careful elimination from the final product of bacterial endotoxins (pyrogens) as well as protein and cell aggregates, through pasteurisation, ultrafiltration and additional chromatography [18] . With this combination of purity and safety advancements, serious adverse events are now rare, even very rare. With equine rabies immunoglobulin (ERIG), anaphylactic shock is reported in less than 1/45, 000 treatments, serum sickness in <0.5% and all-grade adverse events in <5% [3, 15] .

Ammonium sulphate precipitation of the F(ab ) 2 was the main method of purification until the mid-1990s when chromatography was introduced [19] . Examples of current production techniques are for that of the rabies equine immunoglobulin produced by QMSI (Queen Saovabha Memorial Institute, Bangkok) and Favirab (Sanofi-Pasteur) as well as equine snake antivenins [19, 20] . Briefly, the techniques involve hyper-immunisation of the source animal daily for several days and collection of sera 2-4 weeks after the last injection, which enables antibody affinity maturation, resulting in a highly avid, concentrated product, which reduces the co-administered load of contaminant animal proteins [21] .

The purification process often starts with an anion-exchange chromatography step which isolates the immunogenic horse IgG subclass, and removes other immunoglobulins, proteins, cell aggregates and contaminants [19] . Enzymatic cleavage or digestion by low pH pepsin of IgG to Fc and active F(ab ) or F(ab ) 2 regions reduces adverse reactions [17] . A second anion-exchange chromatography excludes the Fc fragments, protein and cell aggregates which result from low pH pepsin digestion, as well as bacterial pyrogens [22] . European guidelines require animal immunosera that are test-negative for pyrogens [23] . A final pasteurisation step involves heat-treatment for 10 h at 60 • C, which destroys viruses, and thermocoagulates excess proteins [20] . High performance liquid chromatography is used to control purity of the final product; usually 90% of the content is covalent F(ab ) 2 , 5% F(ab) fragments, and <0.5% are polymers/aggregates [18] . Importantly, immunoreactivity remains intact after these steps [19] . The final antiviral antibody titre is determined for example by ELISA or seroneutralisation assays for antiviral immunoglobulins. Variations in the production process include application of potassium sulphate for F(ab ) 2 -Fc separation, and precipitation of non-immunoglobulin proteins, caprylic acid precipitation to purify whole IgG (resulting in near total exclusion of albumin, less activation of complement as well as lower total protein/protein aggregates compared to ammonium sulphate precipitation), and centrifugation to eliminate cellular elements and proteins [3, [24] [25] [26] [27] . Preservatives are added to the final products to prevent bacterial and fungal contamination [28] . Refrigeration and avoidance of prolonged storage avoids protein/cell reaggregation and precipitation [28] .

Apart from pasteurisation, steps that neutralise pathogens include low-pH pepsin hydrolysis and high-temperature caprylic acid precipitation, both of which lipolyse enveloped viruses such as Herpes, Sindbis and West Nile viruses; and ultra/nano filtration e.g. using 0.22 m gauge filters, for virus and bacterial removal [4, 29, 30] . Subculturing can confirm bacterial sterility [4] . The WHO, in recognition of some inconsistency in antivenin production quality, has released the 'WHO Guidelines for the Production, Control and Regulation of Snake Antivenom' [31] .

Large relative molecular mass (Mr) bivalent antibodies (IgG and F(ab ) 2 fragments) have a smaller volume of distribution and a longer half-life in the human body than the lower Mr F(ab ) fragments [32] . Both IgG and F(ab ) 2 fragment elimination occurs mainly by formation and elimination of immune complexes (catabolism), whereas F(ab ) is cleared renally [33] .

The longer half-life means that antivenins made from IgG and F(ab ) 2 persist, which reduces rebound symptoms of envenomation associated with F(ab ) antitoxins [34] . Additionally, the preservation of large Mr antivenins in the vascular compartment draws venin out of tissues into the bloodstream to form antivenin-venom complexes, promoting venom clearance [35] . However, recent human pharmacokinetic data suggests that F(ab ) 2 also extravasates into tissues [36, 37] . This tissue penetration may assist with neutralizing viruses infecting various organs.

Cleaving of the Fc fragment, whilst reducing adverse events, possibly reduces it's potentiating effect on generation of natural immunity towards a pathogen or toxin. Fc interaction with antigen-presenting may stimulate and promote development of active immunity towards the exposure [38, 39] .

These features may have implications for the selection of molecule type in the production of polyclonal immunotherapy, particularly for post-exposure prophylaxis against viruses such as Mers-CoV and Ebola. Dosing intervals are longer and therefore dosing regimens more pragmatic with IgG and F(ab ) 2 and IgG also may promote development of host immunity. However, this needs to be balanced against the increased risk of hypersensitivity reactions from the Fc molecule.

Half-time of elimination (t1/2) of the product in the plasma compartment was analyzed after intravenous (IV) administration of F(ab ) 2 against avian influenza A H5N1 in 3 healthy volunteers receiving 1 dose and in 10 healthy volunteers receiving 5 doses [40] . The plasmatic elimination of F(ab ) 2 after one IV infusion had a mean t1/2 of 16.77 h and after 5 infusions 24 h apart, a mean of 10.89 h. These results indicated the persistence of equine F(ab ) 2 in the plasma for the duration of the therapeutic protocol with evidence of a slight accumulation between day 1 and day 5. After the fifth infusion, equine F(ab ) 2 remained detectable by ELISA in plasma, (above > 1 g/mL) for between 3 to 14 days. These results were consistent with another human study of intravenous equine F(ab ) 2 , with a plasma t1/2 of 14.2 h and a later catabolism of F(ab ) 2 with a t1/2 of around 7 days [37] . Thus, the protection induced may continue several days after the end of the treatment protocol, up until the patient's immunity generates host antibodies.

A meta-analysis of seven studies and a Cochrane review each concluded that adrenaline premedication, but not other agents, significantly reduced early adverse reactions [41] . Additionally, early use of adrenaline for anaphylactic reactions post antivenin is effective [42] .

Ovine (sheep-derived) products may be somewhat less reactogenic than equine products, but the latter are more economical to produce given the larger amounts of sera available, and have a longer half-life, reducing requirement of re-administration [43, 44] . Equine antivenin may also have superior antitoxin effects compared to ovine [45] .

There are no recorded cases of viral or prion transmission from equine-derived antitoxins and current processes aim to preserve this safety record [29] . Both initial measures (e.g. donor selection, epidemiological exclusion, quarantine, health status of the animal) and processing of final product reduce the risk of a contaminating virus. Animals are contained within closed flocks or maintained in areas free of insect vectors of certain arboviruses. For example, rattlesnake, viper and digoxin antitoxins are manufactured in South Australia, a region free of prions and many viral pathogens [46] . Source animals may also be vaccinated against local pathogens such as rabies, anthrax and viral equine encephalitides [31] . Molecular diagnostic screening of animals for viruses may be performed [29, 31] . Record-keeping and regular stock inspections contribute to quality control [4] . The rigor of the application of such processes varies by resource availability e.g. in resource-challenged countries where much of the world's antivenin/antitoxin is both required and produced, not all safety measures may be employed [15, 47] . Polyvalent (source animal immunised with more than one venin) polyclonal antivenins have higher rates of adverse reactions than monovalent (source animal immunised to just one venin) polyclonal antivenins, e.g. 24% vs. 9% for Australian produced CSL snake antivenins, due in part to the larger volumes required to be administered for treatment with the polyvalent rather than monovalent polyclonal antibodies [48] .

Clinical studies have demonstrated the safety and efficacy of equine immunoglobulin. These therapies can even be given to pregnant women as no passage of F(ab ) 2 across the placenta is expected and thus no teratogenicity anticipated [49] .

Of 7660 Filipino recipients of F(ab ) 2 equine rabies immunoglobulin (ERIG), (Favirab, Sanofi Pasteur, Lyon, France) only two developed rabies; neither had received post-exposure prophylaxis (PEP) strictly as per the WHO guidelines [50, 51] . Of the151 subjects in this cohort who sustained bites from laboratory-confirmed rabid animals, there were no reported cases of rabies. Of 193 persons bitten by rabid dogs in the Philippines, there was just a single recorded PEP failure; due to location of the bite (on the lip) local ERIG infiltration was difficult, and, against official protocol, she received a mix of intradermal and intramuscular rabies vaccine [52] . These results emphasise the importance of adherence to WHO rabies guidelines in administering PEP.

The safety of current ERIG products has also been demonstrated in clinical trials and post marketing surveillance. Of over 12,000 ERIG recipients in the Philippines, Thailand and India, 0.3 and 1% of recipients developed local reactions and 0.03-3% had systemic reactions [15, [52] [53] [54] . Some of these reactions may have been due to co-administered tetanus toxoid.

A retrospective review of over 70,000 patients in Thailand who received either human rabies immunoglobulin (HRIG) (59.6%) or ERIG (40.4%) demonstrated that 1.83% of ERIG recipients had an adverse events, versus 0.09% of HRIG recipients; however the broad date ranges included ERIG produced both before and after the introduction of modern purification techniques [55] . Serum sickness was reported in 0.72% of ERIG recipients vs. 0.007% of HRIG recipients, and no deaths were reported.

Antivenins are also effective. While there are few randomised prospective controlled human studies, mice studies, retrospective human studies and human case reports provide evidence for effective and safe treatment of life-threatening envenomations and coagulopathy [56, 57] .

Several human reports confirm efficacy of various snake antivenins (Tables 1 and 2 ) [33, [58] [59] [60] [61] [62] .

Efficacy of rattlesnake antivenin has been studied, particularly in the USA, where rattlesnakes predominate; introduction of rattlesnake whole IgG polyvalent crotalid antivenin (ACP) reduced mortality from up to 25% to <0.5% when delivered in a health care facility in the United States [63] . A 10-year retrospective chart review revealed the fractionated Crotalidae polyvalent immune F(ab ) (CroFab ® ) to be more effective at avoiding fasciotomies than the whole IgG product ACP [64] . A 12 year review of CroFab ® revealed a response rate of 77% amongst 24 cases of severe envenomation, including neurotoxicity, whilst in another series of 28 severe envenomations, all responded to CroFab ® [65, 66] . A literature review of controlled and observational studies confirmed the efficacy and safety of CroFab ® [67] . Crofab ® has also been demonstrated to be effective in paediatric patients [68, 69] . However, a South American study demonstrated a locally produced whole IgG antivenin was more effective than two F(ab ) 2 preparations but with more anaphylactoid reactions [70] . Amongst the fractionated products, a comparison of F(ab ) and F(ab ) 2 rattlesnake antivenin demonstrated a significant reduction in late coagulopathy in those who received F(ab ) 2 compared to F(ab ) (29.7% vs. 10.3%) due to the longer half-life of the divalent over the monovalent product [34] . This advantage of F(ab ) 2 over F(ab ) has been demonstrated for other antivenins [71, 72] . Epidemiological data from Europe demonstrates that the introduction of antivenin has resulted in a several-fold decrease in snakebite mortality [73] . Australian snake antivenins have been considered more effective at neutralising the neurotoxic effects than the pro-coagulant effects, and plasma transfusions are recommended along with antivenin [46, 74, 75] . This limitation of current Australian antivenins has been disputed, however [76] .

Safety of snake antivenins has improved with antibody fractionation and modern processing. Whole IgG rattlesnake antivenin (e.g. Antivenin Crotalidae Polyvalent ACP) was associated with a 18-50% frequency of immediate and delayed reactions [77] [78] [79] [80] [81] [82] [83] . This was more than halved after the removal of Fc and implantation of modern purification methods (e.g. Crofab ® ) [ A meta-analysis of CroFab ® revealed an immediate hypersensitivity rate of 8% and serum sickness of 13% [95] , with even lower rates reported in a subsequent study of 340 cases: <2% immediate hypersensitivity reactions and 5% serum sickness [96] . Likewise, the adverse event rate of bothrops antivenin improved from 25 to 82% for ammonium sulphate precipitated whole IgG preparations to 11-28% for caprylic acid precipitated whole IgG and 12-36% for F(ab ) 2 antivenin [26, 28] .

A meta-analysis was conducted by us, the authors, of 30 observational and controlled studies of various snake antivenins, excluding rattlesnake antivenin for which a meta-analysis has been reported above [27, 33, 43, 48, 59, 60, 62, [70] [71] [72] [97] [98] [99] [100] [101] [102] [103] [104] [105] [106] [107] [108] [109] [110] [111] [112] [113] [114] [115] [116] (Table 2) . A simple linear weighting by the sample size of each study was applied. The weighted mean and standard deviation for each set of studies were compared using a formula to isolate the effect of the independent variable (the anti-venin) given to an otherwise similar population distribution. We determined an estimate of the percentage difference with z = 1.96 to give 95% confidence intervals, which are reported below. This analysis demonstrates that F(ab ) and F(ab ) 2 are safer than whole IgG and that there was a reduction in adverse events after introduction of purification techniques in the mid-1990s, with the exception of no reduction in anaphylaxis pre and post 1995 for all types of antivenins, combined.

There are approximately 1.5 million scorpion envenomations annually resulting in about 2600 deaths, from autonomic overstimulation and/or an overwhelming inflammatory response. A randomised controlled trial (RCT) of anti-scorpion F(ab ) 2 in Arizona revealed a significant difference in the following: rapid symptom resolution, midazolam requirement and plasma venom levels, compared to placebo [117] . A meta-analysis of 4 RCTs and 5 observational studies demonstrated effectiveness in new world (American) scorpion envenomations but not in the old world [118] . Adverse events were higher in non-randomised trials, e.g. serum sickness occurred in 57% of those treated in one study, but lasted only 3 days, and antivenin was highly effective, reducing symptom duration from 22 h to 31 min [119] . Adverse events were infrequent in the RCTs (0-2%). However, this meta-analysis included a 2011 Indian ('old world') controlled trial where scorpion F(ab ) 2 antivenin reversed clinical envenomation more effectively than no antivenin [120] . Three other old-world studies not included in the meta-analysis have demonstrated efficacy. An uncontrolled Indian study of scorpion F(ab ) 2 antivenin where 41/48 (85.4%) responded, with one mild reaction [121] ; a prospective case-control series from India of 62 patients under the age of 18 years which demonstrated that dopamine and dobutamine requirement was reduced and recovery was faster in those who received scorpion antivenin [122] and a controlled trial from Saudi Arabia that reported a reduction in mortality from 4-8% to <0.05%, with mild reactions only (13.9%) [123] . In a review of 10 studies, only this Indian uncontrolled trial of scorpion F(ab ) 2 antivenin revealed efficacy, as opposed to whole IgG in all the other trials [124] . However, withdrawal of IgG scorpion antisera in the USA in 2002 with no alternative substituted, resulted in a 5-fold increase in ICU admissions for stings [125] . Saudi Arabian data demonstrated a reduction in deaths from 1.7% to nil, in pulmonary oedema from 11.1% to 1.2% and in cardiac arrest from 7.4% to 0.4% after introduction of scorpion antivenin in 1991 [126] .

Spider antivenin is also effective and safe. A review of whole IgG funnel-web spider antivenin use in Australia reported a complete response in 97% of 75 recipients, with only one local reaction, one case of anaphylaxis and one case of serum sickness [127] . Whole IgG black widow spider (lactodectrus) antivenin reduced durations of symptom and hospitalisation in moderate to severe envenomation in a US review [128, 129] . Intravenous (as compared to intramuscular) administration was associated with high rates of anaphylaxis (e.g. 9% in 1989) and serum sickness (33%) prior to reducing the speed of administration, which reduced the total adverse event rate to less than 3% [130] . Equine derived F(ab ) 2 black widow antivenins have been developed and are being tested [131] . An Australian study of 95 cases of redback spider antivenin F(ab ) 2 yielded 4 cases of anaphylaxis (4.2%) and a serum sickness rate of 9.3% [132] . Overall, polyclonal antibodies used as antidotes are also reportedly safe. A study of 717 patients who received anti-digoxin F(ab ) revealed an allergy rate of 0.8% [133] .

Polyclonal serum therapy is emerging as potentially applicable to a range of viruses for which there are limited therapeutic options.

Highly pathogenic avian influenza (HPAI) viruses such as H5N1 and H7N9 are new targets for polyclonal F(ab ) 2 immunoglobulin therapy. Stockpiles of effective product for prophylaxis and treatment are required in anticipation of epidemics. The neuraminidase inhibitor oseltamivir remains the mainstay of treatment with an overall reduction of mortality risk reported of 49% [134] . Reports of oseltamivir resistance in HPAI H5N1 as well as in seasonal human influenza strains suggests complementary treatment options are necessary [135, 136] .

The World Health Organisation (WHO) recognises the potential role of serum therapy for influenza pandemic planning, given its historical success in infectious outbreaks [137] . Use of convalescent plasma during the 1918 Spanish influenza epidemic of 1918 apparently reduced mortality by 50% [5, 138] . Convalescent plasma has been used in two cases of HPAI H5N1, both of whom survived [139, 140] . Since the first human case of HPAI H5N1 in 1997, the WHO has recorded 718 human infections with 413 deaths as of 26 January 2015, a case fatality rate of 57.5% [141] . All cases have been in Africa, Asia or the Middle East, or travellers returning [142] . Survivors produce demonstrable neutralising antibodies [143] . Whilst there is currently only limited person-to-person transmission, antigenic shift (via reassortment with circulating human viruses) could confer this, setting the stage for a devastating pandemic [144] .

In vivo proof-of-concept studies of equine polyclonal F(ab ) 2 to HPAI H5N1 have been conducted in mice [145] . The product was able to prevent infection after an intranasal HPAI H5N1 challenge. Sero-neutralising assays and haemagglutination inhibition tests confirmed the potent neutralising abilities of equine polyclonal anti-HPAI H5N1 F(ab ) 2 preparations in mice [145] . In another study, four H5N1 avian influenza equine F(ab ) 2 preparations demonstrated cytopathic effect against cultured Madin-Darby canine kidney (MDCK) cells infected with H5N1 and protected mice against lethal challenge, both given prior (prophylaxis) or post (therapeutic) exposure [146] . These studies concur with earlier mice studies of polyclonal antibodies to seasonal influenza A. In one such study, mice were immunised with the M2 antigen of influenza A, anti-influenza IgG was obtained and intravenously injected into other exposed mice leading to 100% survival with high dose (320 micrograms) IgG [147] .

A phase 1 study in 16 healthy young human males aged 21-40 years who received intramuscular polyclonal F(ab ) 2 to HPAI H5N1 yielded no serious adverse events, no changes in blood or urinary parameters, and only one febrile reaction, likely related to the product; there was also evidence of clinical benefit as assessed by seroneutralising and haemagglutination inhibition testing of the subjects' plasma samples [40] . As ethically no placebo-controlled efficacy studies in humans can be performed, further effectiveness data will have to be gathered from compassionate case-based use.

Other fatal avian influenza viruses are emerging in people; in March 2013 the H7N9 avian influenza virus emerged in China and spread to Hong Kong, Taiwan and Malaysia; 718 cases and 413 deaths have been reported to date [148] . Both HPAI H5N1 and H7N9, have demonstrated rare person-to-person transmission [148] [149] [150] [151] . In May 2013, the first case of avian influenza A H6N1 was reported in Taiwan [152] . Three human cases with avian influenza H10N8 were reported in China between December 2013 and February 2014 [153] [154] [155] [156] [157] [158] [159] [160] [161] [162] [163] [164] [165] . Both of these are low pathogenic strains currently, but the potential remains for acquisition of mutations that may increase pathogenicity. Several studies have suggested the potential role of respiratory tract administration of polyclonals to prevent and treat seasonal influenza infection, suggesting a potential for these to be applied to avian influenza strains [156] [157] [158] .

As well as the threat of new avian influenza viruses, the Middle Eastern respiratory syndrome coronavirus (MERS-CoV) that emerged in 2012 has pandemic potential. MERS-CoV causes severe respiratory illness and, sometimes, renal injury [133, 159] . Whilst most cases have occurred in several Middle Eastern countries, particularly Saudi Arabia, cases have been reported from Korea, Europe and North Africa [160, 161] Of the 1368 reported cases of Mers-CoV to WHO by 7 July 2015, 487 have died (35.6%); but a large proportion of cases may go undiagnosed [162] . There appear to be a mix of zoonotic (bat and/or camel) and human sources for transmission; person-to-person transmission has been particularly documented in health-care settings [162] [163] [164] . There is neither a specific treatment nor a licenced vaccine. In a recent study, serum of Egyptian dromedary camels who were seropositive for Mers-CoV was administered to mice infected with Mers-CoV, with Australian camel sero-negative sera serving as a control [165] . Mers-CoV seropositive camel serum given both pre-and post-exposure protected infected mice from weight loss, diminished lung histological changes and accelerated virus clearance. Polyclonal antibodies could provide post-exposure prophylaxis for close contacts as well as a therapeutic strategy for those severely affected by MERS-CoV. One potential setting for the development of an epidemic of either H5N1 or MERS-CoV is the annual HAJJ pilgrimage in Saudi Arabia, where preparations for a potential MERS outbreak are being refined [166] .

Equine polyclonal antibody therapies could also be developed for other widespread and severe neglected tropical diseases e.g. the viral haemorrhagic fevers Crimean-Congo Haemorrhagic Fever, Dengue, Ebola and Marburg; bat-transmitted viruses such as Nipah and Hendra, as well as Lassa virus, West Nile Virus (WNV) and severe acute respiratory syndrome-associated coronavirus (SARS). The WHO has reported 27,705 cases of Ebola with over 11,269 reported deaths in the 2014-2015 West African epidemic [167] .1 A stockpile of polyclonal antibodies may form part of epidemic preparation.

Animal studies exist demonstrating efficacy of polyclonal antibody therapy for various neglected tropical viral diseases. Unimmunised hamsters that received WNV-immune hamster antisera one hour before and 24 h after a WNV challenge were protected from lethal WNV infection [168] . Duck egg polyclonal F(ab ) 2 neutralised Andes virus (the primary agent for pulmonary hantavirus syndrome in South America) in vitro; hamsters given the polyclonal F(ab ) 2 post-exposure had improved survival compared to controls [169] . Polyclonal antibodies against the Marburg and Ebola filoviruses were acquired from non-human primates (NHPs) that survived filovirus challenge and, when given post-exposure, prevented disease and death in virus-challenged NHPs compared to controls [170] . Similar results were obtained from injecting Ebola infected mice with polyclonal sera from E. bola immunised mice [171] . Other studies of mice, monkeys and guinea pigs have demonstrated purified equine whole IgG has prolonged survival against Ebola [172] [173] [174] [175] [176] . Equine F(ab') 2 has been verified to protect both mice and hamsters from development of infection with SARS-CoV infection when given prophylactically and reduced lung viral titres when given therapeutically, compared to controls [177] [178] [179] .

In summary, clinical studies have demonstrated the efficacy of animal-derived polyclonal antibody therapies against various infections, toxins and venoms. The safety of current products has been demonstrated in clinical trials and post marketing surveillance. When WHO guidelines are followed in administering rabies equine polyclonal antibodies, post-exposure prophylaxis is highly effective in averting rabies. Antivenins are also safe and effective for snake and arachnid bites, with available data suggesting a positive effect against scorpion bites. Antidotes are also reportedly safe and effective. Polyclonal antibodies are being developed against viruses with epidemic and pandemic potential for which prophylactic and therapeutic options are currently limited, such as avian influenza and other zoonotic viruses. Preclinical studies as well as phase 1 safety studies against avian H5N1 influenza, are promising and the technology exists to rapidly apply the methods of polyclonal production against a wide range of pathogenic antigens, providing an exciting opportunity to revolutionise the prognosis of both longstanding neglected tropical diseases as well as emerging viral threats to humans.

The authors have no conflict to declare.

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A randomized blinded clinical trial of two antivenoms, prepared by caprylic acid or ammonium sulphate fractionation of IgG, in Bothrops and Porthidium snake bites in Colombia: correlation between safety and biochemical characteristics of antivenoms

Effect of preservatives on IgG aggregation, complement-activating effect and hypotensive activity of horse polyvalent antivenom used in snakebite envenomation

Assessment of the viral safety of antivenoms fractionated from equine plasma

Low pH formulation of whole IgGantivenom: impact on quality, safety, neutralizing potency and viral inactivation

WHO guidelines for the production, control and regulation of snake anitvenom immunoglobulins

Pharmacokinetic-pharmacodynamic relationships of immunoglobulin therapy for envenomation

Clinical safety of a polyvalent F(ab )2 equine antivenom in 223 African snake envenomations: a field trial in Cameroon. VAO (Venin Afrique de l'Ouest) Investigators

Comparison of F(ab )2 versus Fab antivenom for pit viper envenomation: a prospective, blinded, multicenter, randomized clinical trial

Effect of antivenom on venom pharmacokinetics in experimentally envenomed rabbits: toward an optimization of antivenom therapy

Modelling Tityus scorpion venom and antivenom pharmacokinetics. Evidence of active immunoglobulin G's F(ab )2 extrusion mechanism from blood to tissues

Pharmacokinetics of a F(ab )2 scorpion antivenom in healthy human volunteers

Post-exposure treatment of Ebola virus using passive immunotherapy: proposal for a new strategy

Active immunity induced by passive IgG post-exposure protection against ricin

Safety, potential efficacy, and pharmacokinetics of specific polyclonal immunoglobulin F(ab )(2) fragments against avian influenza A (H5N1) in healthy volunteers: a single-centre, randomised, double-blind, placebo-controlled, phase 1 study

Interventions for preventing reactions to snake antivenom

Australian Snakebite Project Investigators. Current use of Australian snake antivenoms and frequency of immediate-type hypersensitivity reactions and anaphylaxis

First clinical experiences with specific sheep Fab fragments in snake bite

A comparison of ovine and equine antivenoms

First clinical experiences with a new ovine Fab Echisocellatus snake bite antivenom in Nigeria: randomized comparative trial with Institute Pasteur Serum (Ipser) Africa antivenom

Twentieth century toxinology and antivenom development in Australia

Snakebite envenoming from a global perspective: towards an integrated approach

Antivenomuse, premedication and early adverse reactions in the management of snake bites in rural Papua New Guinea

Neonatal Fc receptors discriminates and monitors the pathway of native and modified immunoglobulin G in placental endothelial cells

Rabies post-exposure prophylaxis in the Philippines: health status of patients having received purified equine F(ab )2 fragment fabies immunoglobulin (Favirab)

WHO guide for rabies pre and post exposure prophylaxis in humans

Rabies post-exposure prophylaxis with purified equine rabies immunoglobulin: one-year follow-up of patients with laboratory-confirmed category III rabies exposure in the Philippines

Post-exposure prophylaxis for rabies with ERIG and IDRV in children

Safety of equine rabies immune globulin

Sex-and age-related differences in rabies immunoglobulin hypersensitivity

Antivenin therapy in the emergency department

Ability of polyvalent (Crotalidae) antivenin to neutralize myonecrosis, hemorrhage and lethality induced by timber rattlesnake (Crotalus horridus horridus) venom

Efficacy and safety of two whole IgG polyvalent antivenoms, refined by caprylic acid fractionation with or without beta-propiolactone, in the treatment of Bothropsasper bites in Colombia

Early infusion of a purified monospecific F(ab )2 antivenom serum for Bothrops Ianceolatus bites in Martinique

Snake (Bothrops lanceolatus) F(ab )2 antivenom (equine) use in Martinique: safety and efficacy

A controlled clinical trial of a novelantivenom in patients envenomed by Bungarus multicinctus

Clinical trial of an F(ab )2 polyvalent equine antivenom for African snake bites in Benin

North American snake envenomation: diagnosis, treatment, and management

A large single-center experience with treatment of patients with crotalid envenomations: outcomes with and evolution of antivenin therapy

Crotaline Fab antivenom appears to be effective in cases of severe North American pit viper envenomation: an integrative review

Short-term outcomes after Fab antivenom therapy for severe crotaline snakebite

Crotalidae polyvalent immune Fab: in patients with North American crotaline envenomation

Safety and efficacy of crotalidae polyvalent immune Fab in pediatric crotaline envenomations

Utilisation of crotalidae polyvalent immune fab (ovine) for Viperidae envenomations in children

Crotaline snake bite in the Ecuadorian Amazon: randomised double blind comparative trial of three South American polyspecific antivenoms

An open, randomized comparative trial of two antivenoms for the treatment of envenoming by Sri Lankan Russell's viper (Daboia russelii russelii)

First clinical experiences with a new ovine Fab Echisocellatus snake bite antivenom in Nigeria: a randomized comparative trial with Institute Pasteur Serum (IPSER) Africa antivenom

Epidemiology of snakebites in Europe: a systematic review of the literature

Failure of antivenom to improve recovery in Australian snakebite coagulopathy

Clotting factor replacement and recovery from snake venominduced consumptive coagulopathy

ASP Investigators. Efficacy of antivenom against the procoagulant effect of Australian brown snake (Pseudonaja sp.) venom: in vivo and in vitro studies

Snake venom coagulopathy: use and abuse of blood products in the treatment of pit viper envenomation

Poisonous snakebite in central Texas: possible indicators for antivenin treatment

Anaphylaxsis and anaphylactoid reactions

Epidemiology and hospital course of rattlesnake envenomations cared for at a tertiary referral center in Central Arizona

Complications of crotalidae antivenin therapy

Serum sickness following administration of Antivenin (Crotalidae) Polyvalent in 181 cases of presumed rattlesnake envenomation

Does the aggressive use of polyvalent antivenin for rattlesnake bites result in serious acute side effects?

Pediatric snakebites: lessons learned from 114 cases

Efficacy, safety, and use of snake antivenoms in the United States

An epidemiological and clinical study of snake-bites in childhood

A randomized multicenter trial of Crotalinae polyvalent immune Fab (ovine) antivenom for the treatment for Crotaline snakebite in the United States

Immediate and delayed allergic reactions to Crotalidae polyvalent immune Fab (ovine) antivenom

Initial postmarketing experience with crotalidae polyvalent immune Fab for treatment of rattlesnake envenomation

Safety and cost-effectiveness of a clinical protocol implemented to standardize the use of Crotalidae polyvalent immune Fab antivenom at an academic medical centre

Incidence of immediate hypersensitivity reaction and serum sickness following administration of crotalidae polyvalent immune Fab antivenom: a meta-analysis

A randomized multicenter trial of crotalinae polyvalent immune Fab (ovine) antivenom for the treatment for crotaline snakebite in the United States

Affinity-purified, mixed monospecific crotalid antivenom ovine Fab for the treatment of crotalid venom poisoning

Safety and efficacy of crotalidae polyvalent immune Fab in pediatric crotaline envenomations

Incidence of immediate hypersensitivity reaction and serum sickness following administration of crotalidae polyvalent immune Fab antivenom: a meta-analysis

Acute hypersensitivity reactions associated with administration of crotalidae polyvalent immune Fab antivenom

Prediction, prevention, and mechanism of early (anaphylactic) antivenom reactions in victims of snake bites

Evaluation of antivenom therapy in Vipera palaestinae bites

Nigeria-UK EchiTab Study Group. Randomised controlled double-blind non-inferiority trial of two antivenoms for saw-scaled or carpet viper (Echisocellatus) envenoming in Nigeria

Clinical effects and treatment of envenoming by Hoplocephalus spp. snakes in Australia: Australian Snakebite Project (ASP-12)

Tiger snake (Notechisspp) envenoming: Australian Snakebite Project (ASP-13)

Comparative study of the efficacy and safety of two polyvalent, caprylic acid fractionated [IgG and F(ab )2] antivenoms, in Bothropsasper bites in Colombia

A randomised controlled trial of two infusion rates to decrease reactions to antivenom

Adder bites. A report of 68 cases

Clinical aspects of snake bite in the Pacific area

Snake envenomation in children: early reactions frequency at antivenom in patients pretreated with histamine antagonists HI and H2 and hydrocortisone

Evaluation of intravenous immunotherapy with purified F(ab )2 fragments (Viperfav)

Low incidence of early reactions to horse-derived F(ab )(2) antivenom for snakebites in Thailand

Sixteen years of severe Tiger snake (Notechis) envenoming in Perth, Western Australia

Severe snakebites in northern KwaZulu-Natal: treatment modalities and outcomes

Clinical effects of red-bellied black snake (Pseudechis porphyriacus) envenoming and correlation with venom concentrations: Australian Snakebite Project (ASP-11)

clinical study of tolerance and effectiveness of a F(ab )(2) polyvalent antienom for African snake bites in Kindia, Guinea

European viper envenomings: assessment of Viperfav TM and other symptomatic treatments

A controlled clinical trial of a novel antivenom in patients envenomed by Bungarus multicinctus

Short report: treatment of snake envenomations by a new polyvalent antivenom composed of highly purified F(ab)2: results of a clinical trial in northern Cameroon

High rate of immediate systemic hypersensitivity reactions to tiger snake antivenom

Antivenom for critically ill children with neurotoxicity from scorpion stings

Meta-analysis of controlled studies on immunotherapy in severe scorpion envenomation

Scorpion envenomations in young children in central Arizona

Efficacy and safety of scorpion antivenom plus prazosin compared with prazosin alone for venomous scorpion (Mesobuthus tamulus) sting: randomised open label clinical trial

Efficacy of species specific anti-scorpion venom serum (AScVS) against severe, serious scorpion stings (Mesobuthus tamulus concanesisPocock)-an experience from rural hospital in western Maharashtra

Effectiveness of anti scorpion venom for red scorpion envenomation

Treatment of the scorpion envenoming syndrome: 12-years experience with serotherapy

Yes or no for serotherapy in the treatment of scorpion stings and envenomations: a systematic review

Lack of scorpion antivenom leads to increased pediatric ICU admissions

clinical evaluation of the effectiveness of antivenom in scorpion envenomation

Funnelweb spider bite: a systematic review of recorded clinical cases

Clinical presentation and treatment of black widow spider envenomation: a review of 163 cases

A US perspective of symptomatic Latrodectus spp. envenomation and treatment: a National Poison Data System review

Examination of adverse events following black widow antivenom use in California

A randomized, double-blind, placebo-controlled trial of a highly purified equine F(ab)2 antibody black widow spider antivenom

Safety of i.v. administration of redback spider antivenom

Digoxin immune Fab therapy in the management of digitalis intoxication: safety and efficacy results of an observational surveillance study

Effectiveness of antiviral treatment in human influenza A(H5N1) infections: analysis of a Global Patient Registry

Clinical management of human infection with avian influenza A (H5N1) virus

Emergence of oseltamivir resistance: control and management of influenza before, during and after the pandemic

Position paper on collection and use of convalescent plasma or serum as an element in pandemic influenza planning

Logistical feasibility and potential benefits of a population-wide passive immunotherapy program during an influenza pandemic

Treatment with convalescent plasma for Influenza A (H5N1) infection

Successful treatment of avian influenza with convalescent plasma

Cumulative number of confirmed human cases of avian influenza A(H5N1) reported to WHO

Human infection with avian influenza A(H5N1) virus-update

Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinaemia

Prophylactic and therapeutic efficacy of human monoclonal antibodies against H5N1 influenza

Specific polyclonal F(ab )2 neutralize a large panel of highly pathogenic avian influenza A viruses (H5N1) and control infection in mice

Cross clade prophylactic and therapeutic efficacy of polyvalent equine immunoglobulin F(ab')2 against highly pathogenic avian influenza H5N1 in mice

Evaluation of anti-influenza efficiency of polyclonal IgG antibodies specific to the ectodomain of M2 protein of influenza A virus by passive immunization of mice

Influenza at the human-animal interface

Probable person to person transmission of novel avian influenza A (H7N9) virus in Eastern China, 2013: epidemiological investigation

Probable person-to-person transmission of avian influenza A (H5N1)

Probable limited personto-person transmission of highly pathogenic avian influenza A (H5N1) virus in China

Human infection with avian influenza A H6N1 virus: an epidemiological analysis

Emergence in China of human disease due to avian influenza A(H10N8)-cause for concern?

Avian influenza A (H10N8). Factsheet

The Malaysian Insider. China records new H10N8 bird flu death

Prevention and treatment of influenza with hyperimmune bovine colostrum antibody

Compared protective effect of nasal immunoprophylaxis using a new human monoclonal IgM antibody, human polyclonal antibodies, F(ab )2, amantadine, and zanamivir for prophylaxis of influenza A virus pneumonia in mice

Modulation of innate immune responses by influenza-specific ovine polyclonal antibodies used for prophylaxis

Clinical features and viral diagnosis of two cases of infection with Middle East Respiratory Syndrome coronavirus: a report of nosocomial transmission

Updated information on the epidemiology of Middle East respiratory syndrome coronavirus (MERS-CoV) infection and guidance for the public, clinicians, and public health authorities

Middle East respiratory syndrome coronavirus (MERS-CoV)-Republic of Korea. Disease Outbreak News

Transmission and evolution of the Middle East respiratory syndrome coronavirus in Saudi Arabia: a descriptive genomic study

Tracking the transmission and evolution of MERS-CoV

Passive immunotherapy with dromedary immune serum in an experimental animal model for Middle East respiratory syndrome coronavirus infection

The MERS-CoV Scenario Modeling Working Group. Estimating potential incidence of MERS-CoV associated with Hajj Pilgrims to Saudi Arabia

Ebola Situation Report-22

Nipah virus: vaccination and passive protection studies in a hamster model

DNA vaccinegenerated duck polyclonal antibodies as a postexposure prophylactic to prevent hantavirus pulmonary syndrome (HPS)

Postexposure antibody prophylaxis protects nonhuman primates from filovirusdisease

Passive transfer of antibodies protects immunocompetent and imunodeficient mice against lethal Ebola virus infection without complete inhibition of viral replication

Evaluation of immune globulin and recombinant interferon-alpha2b for treatment of experimental Ebola virus infections

Preparation and use of hyperimmune serum for prophylaxis and therapy of Ebola virus infections

Development and study of the properties of immunoglobulin against Ebola fever

The evaluation in hamadryas baboons of the possibility for the specific prevention of Ebola fever

Passive immunization of Ebola virus-infected cynomolgus monkeys with immunoglobulin from hyperimmune horses

Inhibition of infection caused by severe respiratory syndrome-associated coronavirus by equine neutralizing antibody in aged mice

Inhibition of severe acute respiratory syndrome-associated coronavirus infection by equine neutralizing antibody in golden Syrian hamsters

Protection from infection with severe acute respiratory syndrome coronavirus in a Chinese hamster model by equine neutralizing F(ab')2

CécileHerbreteau-Delale (Fab'entench).