- Email: [email protected]
Anti-infective antibodies—Reviving an old paradigm
Vaccine 27S (2009) G33–G37
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Anti-infective antibodies—Reviving an old paradigm Peter Lachmann ∗ Emeritus Sheila Joan Smith Professor of Immunology, Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, United Kingdom
a r t i c l e
i n f o
Article history: Received 20 May 2009 Received in revised form 24 September 2009 Accepted 30 September 2009
Keywords: Antibody therapy Flu Infantile diarrhoea
a b s t r a c t Antibody therapy for infections has a long history and the development of monoclonal antibody technologies, and of increasingly ingenious techniques for making these products, has brought about a major revival in interest. In this paper the field is reviewed with particular emphasis on two topics: the role that antibodies may have in combating pandemic flu; and the prospects for giving, as a food, antibodies derived from the ovalbumin of transgenic chicken eggs, as a prophylactic against diarrhoeal disease. © 2009 Published by Elsevier Ltd.
1. Introduction The first Nobel Prize for medicine was given in 1901 to Emil von Behring for the development of anti-diphtheria serum. Fig. 1 shows a contemporary cartoon where Behring is tapping blood from his horse which the patients are waiting to put into jugs. This may just show a misunderstanding of how antibodies were used at that time or may be – as you will see towards the end of this paper – a very prescient realisation that even oral antibody therapy has great potential. In the period before the introductions of sulphonamides in the 1930s and antibiotics at the end of the Second World War antiinfective antibody therapy was quite widely used. Parrish [1] gives the following list of what was in use at that time: Antitoxic Horse Sera against diphtheria, haemolytic streptococci, Shiga dysentery, tetanus, and gas gangrene. Antibacterial Horse Sera against pneumococci, meningococci and leptospira. Antiviral Sera: Convalescent human sera against measles and yellow fever. Horse antisera against polio, influenza and canine distemper. The horse serum that was widely used at that time was not entirely safe. It could give rise to anaphylaxis due to the formation of IgE antibodies to the horse protein; or to serum sickness due to the formation of IgG antibodies and the formation of immune complexes. It could also give rise to very severe Arthus reactions at
∗ Tel.: +44 01223 766242; fax: +44 01223 766244. E-mail address: [email protected]. 0264-410X/$ – see front matter © 2009 Published by Elsevier Ltd. doi:10.1016/j.vaccine.2009.09.137
the injection sites, especially where large volumes of serum were injected, as was the case when treating pneumococcal pneumonia. These Arthus reactions could cause extensive muscle destruction of a kind that is no longer seen now that Arthus reactions are just little red lumps seen when small amounts of antigen are injected into the skin. For these reasons, horse serum and immunoglobulins fractionated from it have largely gone out of use, although some horse anti-venoms to snake venom may still be used. In the 1930s human immunoglobulins from convalescent or immunised subjects came into use, and indeed continue to be used. The discovery of monoclonal antibodies in the 1980s started a new era in antibody therapy with the ability to make very large quantities of mono-specific antibodies which could be derived from animals and subsequently humanised by molecular biology or indeed derived from human lymphocytes. Even more recently, as will be discussed with regard to the new broad spectrum anti-flu antibody, it has become possible to make monoclonal antibodies not from a library of antibody molecules found in vivo but from phage display libraries which may represent antibodies that are entirely novel and have not been made in response to the antigen by a host. During the 21st century increasingly ingenious techniques are being developed to make recombinant monoclonal antibodies, not only using human cells in tissue culture but making antibodies from transgenic plants, from silk worm larvae, and in the milk of transgenic animals. Very recently, (as will be discussed below) the use of transgenic chickens who make antibodies in the ovalbumin of their eggs, have been described and this is a particularly attractive technique. However, none of these very recent techniques have yet given rise to products licensed for clinical use although it is to be hoped that they soon will.
G34
P. Lachmann / Vaccine 27S (2009) G33–G37
Fig. 1. “The Pharmacy of the Future 1901”.
Antibody therapies currently in use against infections include the following: Pooled human immunoglobulins for antibody deficiency states are widely used. Their use in travellers for the prophylaxis of (particularly) Hepatitis A is now disappearing as the majority are vaccinated against the virus. Pathogen specific polyclonal human immunoglobulins are used in selected subjects exposed to measles, CMV, varicella, hepatitis B, rabies, vaccinia, rubella, ebola and other haemorrhagic fevers. Antitoxic antibodies for tetanus and diphtheria are used in the unvaccinated and antibodies to snake venoms are also still used. Monoclonal antibodies of a subclass that does not react with human Fc receptors are used to treat RSV without giving rise to immunopathology. The present emphasis is on virus infections because antiviral drugs are much less developed than antibiotics, although because of the increasing problem of antibiotic resistance in bacteria, this situation may not last and antibacterial antibodies may become increasingly used again.
gen only in the context of MHC antigens which the great majority of viruses do not carry on their membranes. In the case of retroviruses, which contain reverse transcriptase whose presence is necessary to achieve infection, lysis of the virion by antibody and complement or indeed by complement alone can also produce sterilising immunity. Some viruses have acquired the capacity to subvert neutralisation by antibody. The most spectacular example of this is with Dengue where antibodies, if they fail to achieve full neutralisation, may enhance infection by several orders of magnitude by facilitating entry of the virus into macrophages through Fc receptors. This is the mechanism underlying Dengue Haemorrhagic Fever. Immunopathology caused by antibody enhancement is also seen with RSV and HIV, though to a much smaller extent. However, for RSV where monoclonal antibodies are used, they are made of a sub-type which does not react with the appropriate macrophage Fc receptors. With many other viruses, including influenza, there is no evidence of antibody mediated immunopathology.
3. Passive immunisation and flu 2. Immunity to virus infections Natural immunity to viruses is mediated partly by the innate immune system through the secretion of interferon and sometimes by direct interaction with the complement system. Some viruses also directly activate Toll-like receptors. The adaptive immune response to natural infection with viruses is usually by killing of virus infected cells using either cytotoxic T-cells or antibodies and complement or antibodies and killer cells. Antibody prevents the spread of the virus within the body and prevents reinfection. However, vaccination with viruses and prophylactic antibody therapy given to the uninfected aims at sterilising immunity which prevents the virus infecting cells in the first place. This is achieved only by antibodies and antibodies alone, since T-cells can see anti-
The recent concerns about pandemic flu—the highly virulent H5N1 bird flu which transmits from birds to humans where it causes high mortality, but so far at least, does not transmit efficiently between humans; and the very recent H1N1 swine flu which appears to transmit efficiently but is so far not very virulent, have brought back into focus the possibility of using passive immunotherapy. This is particularly the case because the H5N1 virus rapidly develops resistance to the current antiviral drugs and it is unclear how readily the new swine flu virus will do the same. Possible sources of anti-flu antibody for passive immunotherapy include immunoglobulins from convalescent human plasma or from immunised volunteers; or human monoclonal antibodies. The latter, made from the lymphocytes of convalescent patients, have
P. Lachmann / Vaccine 27S (2009) G33–G37
G35
Fig. 2. In vivo efficacy of equine Fab’2 anti-H5N1 (from [4]).
been used experimentally in mice and shown to be effective [2]. However, to prevent rapid development of resistance it is probable that three separate antibody specificities would be needed. A major problem of the use of such antibodies is that their production in quantity remains very expensive and takes time. It would also be possible to immunise volunteers with the existing experimental vaccines and to make immunoglobulins from their plasma by the standard procedures that were used in the past for making anti-Rhesus D immunoglobulin to prevent haemolytic disease of the newborn. This is estimated to cost about £450 per litre by the Blood Transfusion Service. In the United Kingdom there has been marked reluctance to embark on this route with H5N1 because of the concerns – probably exaggerated – of transmitting variant Creutzfeld Jacob disease. Passive antibodies could be used, for example in H5N1 flu, to protect those who have to come into contact with infected birds, or their family contacts, and is also valuable to treat early infection. There is some suggestion that antibodies, even those that do not fully neutralise this very virulent virus, may be able to prevent the cleavage of the haemagglutinin at extra-respiratory sites and thereby prevent the spread into the gut and into the brain. There is a further possible advantage of giving antibody therapy if the patients subsequently become infected. This derives from the work of Renegar et al. [3] who found that IgG antibodies against flu protect the lung from disease but do not protect the nose from infection in mice, which only polymeric IgA was able to prevent. This raises the possibility that infection after giving passive IgG antibody would provide a form of surrogate vaccination whereby the subject becomes actively immune without developing the disease. It is quite plausible that the current, killed, vaccines against seasonal flu that generate largely IgG antibodies act in a similar way. In a mouse model, it has been clearly shown by Lu et al. [4] that a F(ab)2 horse anti-H5N1 protects both in tissue culture and protects mice experimentally infected (Fig. 2). However, the whole field of passive immunotherapy to flu has very recently been transformed by the development of monoclonal antibodies from phage display which react with the fusion site on influenza viruses and that prevent infection by a wide variety of influenza viruses, including H5N1 and H1N1. Two groups have reported on this topic very recently: Sui et al. [5]; and, independently, Dr Uytdehaag from Crucell who presented his findings at this symposium. Their findings are revolutionary and the monoclonal antibodies both prevent infection in vitro and prevent death in vivo in an extremely impressive fashion. It would seem highly
Fig. 3. Immunodominant peptides are also recognised in normal subjects. HIV-1 gp41 peptides recognized by sera from 13 HIV-1 seropositive individuals (a) or four laboratory personnel (b). Cumulative totals of antibody determinations by radioimmunoassay are displayed (from [6]).
desirable that these antibodies are soon made in large quantities as an additional weapon either against H5N1 or against H1N1. However, as is the problem with all monoclonal antibodies, their production in large quantities is expensive and it is to be hoped that some of the cheaper and more adventurous techniques that were discussed earlier can be applied to these highly promising antibodies. 4. Why are these antibodies not made after natural infection? This is of course a question of great practical importance and it seems likely that viruses have evolved strategies that focus the immune response of their hosts on epitopes that are not fully preventive of infection as a subversion mechanism. Indeed, all successful pathogens have developed methods of subverting the immune response and the variety is very impressive. This particular form of subversion is known with HIV. Fig. 3, taken from Davis et al. [6], shows antibody responses to peptides derived from HIV gp41 where it can be seen that those peptides that elicit particularly high responses in HIV positive individuals also give some response in HIV negative lab personnel, suggesting that these are secondary responses to epitopes that may be shared with other viruses. Fig. 4 shows a rather primitive data scanning performed in 1990 [6] and shows that for one of the major peptide responses to Env61 there are indeed a wide variety of rather similar sequences in a whole variety of viruses. It therefore seems likely that the immune response is being deviated in this way to the secondary responses in preventing response to other epitopes. It is possible, in theory at least, to avoid this form of subversion by using a technique known as “cascade immunisation”. Cascade immunisation is a technique to avoid antigenic competition by stronger epitopes using passive antibody to the dominant epitope. An example is shown in Fig. 5 taken from a paper by Taussig and Lachmann [7], where we were trying to make antibodies to the Fd region of intact immunoglobulin. These antibodies are not made by immunising with whole IgG but are made either if the animal
G36
P. Lachmann / Vaccine 27S (2009) G33–G37
Fig. 4. Other viruses sharing sequence with Env61. Antigens having five or more amino acids, in a sequence of seven, identical with peptide Env61 (from [6]).
is made tolerant to Fc or to a very similar extent by giving passive Fc antibody shortly after giving the antigen. If used for flu the idea would be to immunise a naive subject and take antibodies at the height of the immune response. A second naive subject is then immunised and given the first antibodies an hour or so after the injection at an adequate dose, and a further set of antibodies will be made. If these still do not contain the desired antibodies, the procedure is repeated, immunising a third naive subject and giving them a mixture of antibodies from the first immunisations. In principle this procedure can be continued ad infinitum or until no further new antibodies are made. It is clearly not a practical method of immunising human populations but it might be a very interesting experimental way of investigating whether it is possible to raise polyclonal antibodies to the fusion site of flu. If this is possible in human volunteers then fully human monoclonal antibodies could be made from their lymphocytes as was done with lymphocytes of patients convalescent from H5N1 infection [2]. I would finally like to consider a slightly different topic which is the use of passive antibody, not as a biopharmaceutical but as a prophylactic food. This, again, is not a new idea and it is several decades since experiments were done with immunising cows with gut pathogens in the hope that their milk would provide pas-
sive protection when it is fed. This was never successful largely because adequate antibody levels were found only in colostrum which is produced only briefly, and of course every cow that makes the antibody has to be immunised. More recently transgenic rabbits, goats and cows have been made that secrete antibodies in the milk but this has turned out to be very expensive and such products have so far only been used as biopharmaceuticals. However, a major advance in this field was introduced from the laboratory of Dr. Helen Sang [8] who generated transgenic hens with oviduct specific expression of therapeutic proteins. This is an extremely attractive technique since these transgenes are inherited stably from chicken to chicken and the breeding of transgenic chickens is very much easier than that of transgenic mammals. It is worth remembering that about 25 billion chicks are hatched each year and a very large number of these are produced by a very small number of companies. Scale-up could therefore be very quick. It would be possible, in addition, to use the remarkable properties of camelid antibodies for oral administration from transgenic ovalbumin. Camelid antibodies have their specificity directed entirely by their heavy chain, making their engineering much easier, but also have the remarkable property of being very heat stable [9] so that a transgenic egg could be lightly boiled to get rid of bac-
Fig. 5. Using passive anti-Fc to bring out anti-Fd response to intact IgG (from [7]).
P. Lachmann / Vaccine 27S (2009) G33–G37
G37
Conflict of Interest The authors state that they have no conflict of interest. References
Fig. 6. “Immunity for breakfast?” (from [10]).
teria without destroying the antibodies. This technique, therefore, would have a promise of making, relatively easily, egg whites containing antibodies to verotoxin or to shigatoxin or to rotavirus which could be fed as food to children in sub-Saharan Africa where the deaths due to such infections are still a major problem. Proof of concept for the use of antibodies to protect mucosal surfaces already exists. Van der Vaart et al. [10] used fragments of llama antibodies against rotavirus to protect mice in vivo and Nilsson et al. [11] used avian antibodies to Pseudomonas aeruginosa flaggellin in children with cystic fibrosis to protect against infection with this organism. Regulatory problems of giving antibodies as food rather than as a drug should also be much less and it is to be hoped that this technique will be developed. However, obtaining financial support for this sort of project is not as easy as it should be. The project was discussed in The Scientist [12] with a rather attractive cartoon which forms a suitable epilogue to this paper (Fig. 6).
[1] Parrish HJ. Bacterial and viral diseases. Livingstone: Edinburgh; 1948. [2] Simmons CP, Bernasconi NL, Suguitan AL, Mills K, Ward JM, Chau NV, et al. Prophylactic and therapeutic efficacy of human monoclonal antibodies against H5N1 influenza. PLoS Med 2007;4(5):e178. [3] Renegar KP, Small Jr PA, Boykins LG, Wright PF. Role of IgA versus IgG in the control of influenza viral infection in the murine respiratory tract. J Immunol 2004;173:1978–86. [4] Lu J, Guo Z, Pan X, Wang G, Zhang D, Li Y, et al. Passive immunotherapy for influenza A H5N1 virus infection with equine hyperimmune globulin F(ab’)2 in mice. Respir Res 2006;7(1):43–9. [5] Sui J, Hwang WC, Perez S, Wei G, Aird D, Chen L-M, et al. Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol 2009;16 [Epub 2009 Feb 22]. [6] Davis D, Chaudhri B, Stephens DM, Carne CA, Willers C, Lachmann PJ. The immunodominance of epitopes within the transmembrane protein (gp41) of human immunodeficiency virus type 1 may be determined by the host’s previous exposure to similar epitopes on unrelated antigens. J Gen Virol 1990;71:1975–83. [7] Taussig MJ, Lachmann PJ. Studies on antigenic competition. II. Abolition of antigenic competition by antibody against or tolerance to the dominant antigen: a model for antigenic competition. Immunology 1972;22:185–97. [8] Lillico SG, Sherman A, McGrew MJ, Robertson CD, Smith J, Haslam C, et al. Oviduct-specific expression of two therapeutic proteins in transgenic hens. PNAS 2007;104(6):1771–6. [9] Harmsen MM, De Haard HJ. Properties, production, and applications of camelid single-domain antibody fragments. Appl Microbiol Biotechnol 2007;77(1):13–22. [10] van der Vaart JM, Pant N, Wolvers D, Bezemer S, Hermans PW, Bellamy K, Sarker SA, van der Logt CP, Svensson L, Verrips CT, Hammarstrom L, van Klinken BJ. Reduction in morbidity of rotavirus induced diarrhoea in mice by yeast produced monovalent llama-derived antibody fragments. Vaccine 2006;24(19):4130–7. [11] Nilsson E, Amini A, Wretlind B, Larsson A. Pseudomonas aeruginosa infections are prevented in cystic fibrosis patients by avian antibodies binding Pseudomonas aeruginosa flagellin. J Chromatogr B: Anal Technol Biomed Life Sci 2007;856(1–2):75–80. [12] Ganguli I. Immunity for breakfast? The Scientist 2008;22(March).
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Anti-infective antibodies—Reviving an old paradigm Peter Lachmann ∗ Emeritus Sheila Joan Smith Professor of Immunology, Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, United Kingdom
a r t i c l e
i n f o
Article history: Received 20 May 2009 Received in revised form 24 September 2009 Accepted 30 September 2009
Keywords: Antibody therapy Flu Infantile diarrhoea
a b s t r a c t Antibody therapy for infections has a long history and the development of monoclonal antibody technologies, and of increasingly ingenious techniques for making these products, has brought about a major revival in interest. In this paper the field is reviewed with particular emphasis on two topics: the role that antibodies may have in combating pandemic flu; and the prospects for giving, as a food, antibodies derived from the ovalbumin of transgenic chicken eggs, as a prophylactic against diarrhoeal disease. © 2009 Published by Elsevier Ltd.
1. Introduction The first Nobel Prize for medicine was given in 1901 to Emil von Behring for the development of anti-diphtheria serum. Fig. 1 shows a contemporary cartoon where Behring is tapping blood from his horse which the patients are waiting to put into jugs. This may just show a misunderstanding of how antibodies were used at that time or may be – as you will see towards the end of this paper – a very prescient realisation that even oral antibody therapy has great potential. In the period before the introductions of sulphonamides in the 1930s and antibiotics at the end of the Second World War antiinfective antibody therapy was quite widely used. Parrish [1] gives the following list of what was in use at that time: Antitoxic Horse Sera against diphtheria, haemolytic streptococci, Shiga dysentery, tetanus, and gas gangrene. Antibacterial Horse Sera against pneumococci, meningococci and leptospira. Antiviral Sera: Convalescent human sera against measles and yellow fever. Horse antisera against polio, influenza and canine distemper. The horse serum that was widely used at that time was not entirely safe. It could give rise to anaphylaxis due to the formation of IgE antibodies to the horse protein; or to serum sickness due to the formation of IgG antibodies and the formation of immune complexes. It could also give rise to very severe Arthus reactions at
∗ Tel.: +44 01223 766242; fax: +44 01223 766244. E-mail address: [email protected]. 0264-410X/$ – see front matter © 2009 Published by Elsevier Ltd. doi:10.1016/j.vaccine.2009.09.137
the injection sites, especially where large volumes of serum were injected, as was the case when treating pneumococcal pneumonia. These Arthus reactions could cause extensive muscle destruction of a kind that is no longer seen now that Arthus reactions are just little red lumps seen when small amounts of antigen are injected into the skin. For these reasons, horse serum and immunoglobulins fractionated from it have largely gone out of use, although some horse anti-venoms to snake venom may still be used. In the 1930s human immunoglobulins from convalescent or immunised subjects came into use, and indeed continue to be used. The discovery of monoclonal antibodies in the 1980s started a new era in antibody therapy with the ability to make very large quantities of mono-specific antibodies which could be derived from animals and subsequently humanised by molecular biology or indeed derived from human lymphocytes. Even more recently, as will be discussed with regard to the new broad spectrum anti-flu antibody, it has become possible to make monoclonal antibodies not from a library of antibody molecules found in vivo but from phage display libraries which may represent antibodies that are entirely novel and have not been made in response to the antigen by a host. During the 21st century increasingly ingenious techniques are being developed to make recombinant monoclonal antibodies, not only using human cells in tissue culture but making antibodies from transgenic plants, from silk worm larvae, and in the milk of transgenic animals. Very recently, (as will be discussed below) the use of transgenic chickens who make antibodies in the ovalbumin of their eggs, have been described and this is a particularly attractive technique. However, none of these very recent techniques have yet given rise to products licensed for clinical use although it is to be hoped that they soon will.
G34
P. Lachmann / Vaccine 27S (2009) G33–G37
Fig. 1. “The Pharmacy of the Future 1901”.
Antibody therapies currently in use against infections include the following: Pooled human immunoglobulins for antibody deficiency states are widely used. Their use in travellers for the prophylaxis of (particularly) Hepatitis A is now disappearing as the majority are vaccinated against the virus. Pathogen specific polyclonal human immunoglobulins are used in selected subjects exposed to measles, CMV, varicella, hepatitis B, rabies, vaccinia, rubella, ebola and other haemorrhagic fevers. Antitoxic antibodies for tetanus and diphtheria are used in the unvaccinated and antibodies to snake venoms are also still used. Monoclonal antibodies of a subclass that does not react with human Fc receptors are used to treat RSV without giving rise to immunopathology. The present emphasis is on virus infections because antiviral drugs are much less developed than antibiotics, although because of the increasing problem of antibiotic resistance in bacteria, this situation may not last and antibacterial antibodies may become increasingly used again.
gen only in the context of MHC antigens which the great majority of viruses do not carry on their membranes. In the case of retroviruses, which contain reverse transcriptase whose presence is necessary to achieve infection, lysis of the virion by antibody and complement or indeed by complement alone can also produce sterilising immunity. Some viruses have acquired the capacity to subvert neutralisation by antibody. The most spectacular example of this is with Dengue where antibodies, if they fail to achieve full neutralisation, may enhance infection by several orders of magnitude by facilitating entry of the virus into macrophages through Fc receptors. This is the mechanism underlying Dengue Haemorrhagic Fever. Immunopathology caused by antibody enhancement is also seen with RSV and HIV, though to a much smaller extent. However, for RSV where monoclonal antibodies are used, they are made of a sub-type which does not react with the appropriate macrophage Fc receptors. With many other viruses, including influenza, there is no evidence of antibody mediated immunopathology.
3. Passive immunisation and flu 2. Immunity to virus infections Natural immunity to viruses is mediated partly by the innate immune system through the secretion of interferon and sometimes by direct interaction with the complement system. Some viruses also directly activate Toll-like receptors. The adaptive immune response to natural infection with viruses is usually by killing of virus infected cells using either cytotoxic T-cells or antibodies and complement or antibodies and killer cells. Antibody prevents the spread of the virus within the body and prevents reinfection. However, vaccination with viruses and prophylactic antibody therapy given to the uninfected aims at sterilising immunity which prevents the virus infecting cells in the first place. This is achieved only by antibodies and antibodies alone, since T-cells can see anti-
The recent concerns about pandemic flu—the highly virulent H5N1 bird flu which transmits from birds to humans where it causes high mortality, but so far at least, does not transmit efficiently between humans; and the very recent H1N1 swine flu which appears to transmit efficiently but is so far not very virulent, have brought back into focus the possibility of using passive immunotherapy. This is particularly the case because the H5N1 virus rapidly develops resistance to the current antiviral drugs and it is unclear how readily the new swine flu virus will do the same. Possible sources of anti-flu antibody for passive immunotherapy include immunoglobulins from convalescent human plasma or from immunised volunteers; or human monoclonal antibodies. The latter, made from the lymphocytes of convalescent patients, have
P. Lachmann / Vaccine 27S (2009) G33–G37
G35
Fig. 2. In vivo efficacy of equine Fab’2 anti-H5N1 (from [4]).
been used experimentally in mice and shown to be effective [2]. However, to prevent rapid development of resistance it is probable that three separate antibody specificities would be needed. A major problem of the use of such antibodies is that their production in quantity remains very expensive and takes time. It would also be possible to immunise volunteers with the existing experimental vaccines and to make immunoglobulins from their plasma by the standard procedures that were used in the past for making anti-Rhesus D immunoglobulin to prevent haemolytic disease of the newborn. This is estimated to cost about £450 per litre by the Blood Transfusion Service. In the United Kingdom there has been marked reluctance to embark on this route with H5N1 because of the concerns – probably exaggerated – of transmitting variant Creutzfeld Jacob disease. Passive antibodies could be used, for example in H5N1 flu, to protect those who have to come into contact with infected birds, or their family contacts, and is also valuable to treat early infection. There is some suggestion that antibodies, even those that do not fully neutralise this very virulent virus, may be able to prevent the cleavage of the haemagglutinin at extra-respiratory sites and thereby prevent the spread into the gut and into the brain. There is a further possible advantage of giving antibody therapy if the patients subsequently become infected. This derives from the work of Renegar et al. [3] who found that IgG antibodies against flu protect the lung from disease but do not protect the nose from infection in mice, which only polymeric IgA was able to prevent. This raises the possibility that infection after giving passive IgG antibody would provide a form of surrogate vaccination whereby the subject becomes actively immune without developing the disease. It is quite plausible that the current, killed, vaccines against seasonal flu that generate largely IgG antibodies act in a similar way. In a mouse model, it has been clearly shown by Lu et al. [4] that a F(ab)2 horse anti-H5N1 protects both in tissue culture and protects mice experimentally infected (Fig. 2). However, the whole field of passive immunotherapy to flu has very recently been transformed by the development of monoclonal antibodies from phage display which react with the fusion site on influenza viruses and that prevent infection by a wide variety of influenza viruses, including H5N1 and H1N1. Two groups have reported on this topic very recently: Sui et al. [5]; and, independently, Dr Uytdehaag from Crucell who presented his findings at this symposium. Their findings are revolutionary and the monoclonal antibodies both prevent infection in vitro and prevent death in vivo in an extremely impressive fashion. It would seem highly
Fig. 3. Immunodominant peptides are also recognised in normal subjects. HIV-1 gp41 peptides recognized by sera from 13 HIV-1 seropositive individuals (a) or four laboratory personnel (b). Cumulative totals of antibody determinations by radioimmunoassay are displayed (from [6]).
desirable that these antibodies are soon made in large quantities as an additional weapon either against H5N1 or against H1N1. However, as is the problem with all monoclonal antibodies, their production in large quantities is expensive and it is to be hoped that some of the cheaper and more adventurous techniques that were discussed earlier can be applied to these highly promising antibodies. 4. Why are these antibodies not made after natural infection? This is of course a question of great practical importance and it seems likely that viruses have evolved strategies that focus the immune response of their hosts on epitopes that are not fully preventive of infection as a subversion mechanism. Indeed, all successful pathogens have developed methods of subverting the immune response and the variety is very impressive. This particular form of subversion is known with HIV. Fig. 3, taken from Davis et al. [6], shows antibody responses to peptides derived from HIV gp41 where it can be seen that those peptides that elicit particularly high responses in HIV positive individuals also give some response in HIV negative lab personnel, suggesting that these are secondary responses to epitopes that may be shared with other viruses. Fig. 4 shows a rather primitive data scanning performed in 1990 [6] and shows that for one of the major peptide responses to Env61 there are indeed a wide variety of rather similar sequences in a whole variety of viruses. It therefore seems likely that the immune response is being deviated in this way to the secondary responses in preventing response to other epitopes. It is possible, in theory at least, to avoid this form of subversion by using a technique known as “cascade immunisation”. Cascade immunisation is a technique to avoid antigenic competition by stronger epitopes using passive antibody to the dominant epitope. An example is shown in Fig. 5 taken from a paper by Taussig and Lachmann [7], where we were trying to make antibodies to the Fd region of intact immunoglobulin. These antibodies are not made by immunising with whole IgG but are made either if the animal
G36
P. Lachmann / Vaccine 27S (2009) G33–G37
Fig. 4. Other viruses sharing sequence with Env61. Antigens having five or more amino acids, in a sequence of seven, identical with peptide Env61 (from [6]).
is made tolerant to Fc or to a very similar extent by giving passive Fc antibody shortly after giving the antigen. If used for flu the idea would be to immunise a naive subject and take antibodies at the height of the immune response. A second naive subject is then immunised and given the first antibodies an hour or so after the injection at an adequate dose, and a further set of antibodies will be made. If these still do not contain the desired antibodies, the procedure is repeated, immunising a third naive subject and giving them a mixture of antibodies from the first immunisations. In principle this procedure can be continued ad infinitum or until no further new antibodies are made. It is clearly not a practical method of immunising human populations but it might be a very interesting experimental way of investigating whether it is possible to raise polyclonal antibodies to the fusion site of flu. If this is possible in human volunteers then fully human monoclonal antibodies could be made from their lymphocytes as was done with lymphocytes of patients convalescent from H5N1 infection [2]. I would finally like to consider a slightly different topic which is the use of passive antibody, not as a biopharmaceutical but as a prophylactic food. This, again, is not a new idea and it is several decades since experiments were done with immunising cows with gut pathogens in the hope that their milk would provide pas-
sive protection when it is fed. This was never successful largely because adequate antibody levels were found only in colostrum which is produced only briefly, and of course every cow that makes the antibody has to be immunised. More recently transgenic rabbits, goats and cows have been made that secrete antibodies in the milk but this has turned out to be very expensive and such products have so far only been used as biopharmaceuticals. However, a major advance in this field was introduced from the laboratory of Dr. Helen Sang [8] who generated transgenic hens with oviduct specific expression of therapeutic proteins. This is an extremely attractive technique since these transgenes are inherited stably from chicken to chicken and the breeding of transgenic chickens is very much easier than that of transgenic mammals. It is worth remembering that about 25 billion chicks are hatched each year and a very large number of these are produced by a very small number of companies. Scale-up could therefore be very quick. It would be possible, in addition, to use the remarkable properties of camelid antibodies for oral administration from transgenic ovalbumin. Camelid antibodies have their specificity directed entirely by their heavy chain, making their engineering much easier, but also have the remarkable property of being very heat stable [9] so that a transgenic egg could be lightly boiled to get rid of bac-
Fig. 5. Using passive anti-Fc to bring out anti-Fd response to intact IgG (from [7]).
P. Lachmann / Vaccine 27S (2009) G33–G37
G37
Conflict of Interest The authors state that they have no conflict of interest. References
Fig. 6. “Immunity for breakfast?” (from [10]).
teria without destroying the antibodies. This technique, therefore, would have a promise of making, relatively easily, egg whites containing antibodies to verotoxin or to shigatoxin or to rotavirus which could be fed as food to children in sub-Saharan Africa where the deaths due to such infections are still a major problem. Proof of concept for the use of antibodies to protect mucosal surfaces already exists. Van der Vaart et al. [10] used fragments of llama antibodies against rotavirus to protect mice in vivo and Nilsson et al. [11] used avian antibodies to Pseudomonas aeruginosa flaggellin in children with cystic fibrosis to protect against infection with this organism. Regulatory problems of giving antibodies as food rather than as a drug should also be much less and it is to be hoped that this technique will be developed. However, obtaining financial support for this sort of project is not as easy as it should be. The project was discussed in The Scientist [12] with a rather attractive cartoon which forms a suitable epilogue to this paper (Fig. 6).
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