Monoclonal antibodies against micro-organisms

Monoclonal

antibodies

against micro-organisms

T. Lehner of Immunology, United Medical and Dental School of Guy’s and St Thomas’ Hospital, London, UK.

Department

Current

Opinion

in Immunology

Introduction Since the discovery of monoclonal antibodies (mAb) (Kohler and M&stein, Nuture 1975, 256:495-~496),~ a plethora of mAb have been generated to micro-or@isms and their structural components. Antibodies against microbial determinants have been broadly used in four areas: (1) (2) (3) (4)

detection, identiiication, classification and diagnosis of micro-organisms; characterization of structurally and functionally defmed antigenic determinants; isolation and purification of microbial glycoproteins, and protection against microbial infection.

Over 1000 papers were published on this subject during the year 1987-1988 and clearly the task of reviewing the literature is too demanding for a brief review. Fortunately, a general review is not required as there is a steady stream of reviews and books on mAb. Instead, a limited selection of topics will be surveyed which are either of immediate importance or have not received adequate attention, namely: (1) (2)

(3)

detection and characterization of human immunodeficiency virus (HIV) epitopes by ti; prevention of microbial infection by central (vascular) and peripheral (mucosal) immunization with mAb, and human mAb to micro-organisms.

Detection

and characterization

of HIV epitopes

by mAb With the isolation of the retrovirus responsible for the acquired immune deficiency syndrome (AIDS) there is a concerted international effort to produce an effective vaccine for protection against infection with HIV. The virus has now been cloned and there are three genes essential

1989, 1:462-466

for virus replication. These code for the internal structural proteins (gag> for the reverse transcriptase (pal) and for the outer envelope glycoprotein (env) of the virus. The latter codes for gp160 which consists of the extracellular gp120 and transmembrane gp41, all of which are candidates for immunization. mAb to gp160 have been produced recently [l ] and these recognize a deglycosylated polypeptide of 900. The mAb recognize an epitope in gp120 of the precursor gpl60 and not of gp41 of HIV-l. The mAb does not recognize HIV-2. As the infectivity of HIV-l is not affected by the presence of the mAb, it does not have neutralizQ activity. The mAb can be used to detect gpl60 in cells transfected with the env gene of HIV-l [2]. It has also been readily applied to purification of gpl60 from crude HIV-1 infected cells by affinity chromatography. mAb to recombinant gpll0 and gp41 have also been raised recently [3] and all of these are of the im munoglobulin (Ig) Gl subclass. Five mAb recognize the central portion of gpll0 (amino acids 279-472) and four of these are capable of neutralization of HIV-l by infection inhibition and syncytial inhibition. One of the two mAb to gp41 also inhibits syncytial formation. It is likely that these mAb recognize a single neutralization epitope of HIV-l which should be capable of precise definition. mAb against a septapeptide derived from gp41 showed cross-reactivity not only with the native HIV-l but also with a non-polymorphic human leukocyte antigen (HIA) class II on human B cell lines [4]. Indeed, a homologous region of five amino acids was identified at the carboxy terminus of HIV-l envelope protein and the amino-terminus of HLA class II p chains which were highly conserved among different DR and DQ alleles and different isolates of HIV-l. This observation was corroborated with the Linding that 36% of sera from patients with AIDS contained anti-class II antibodies. The possibility is raised that HIV-l infected patients may develop anti-self HLAclass II antibodies which may trigger an autoimmune process that could further contribute to the manifestations of AIDS. Such antibodies may interfere with the normal cellular interactions between CD4+ T cells and class II bearing antigen-presenting cells. Indeed, the mAb can

Abbreviations AIDS-acquired immune deficiency syndrome; HIV--human immunodeficiency virus; HU-human leukocyte antigen; HSV-herpes simplex virus; Ig-immunoglobulin; LPS-lipopolysaccharide; m&monoclonal antibody; PWM-pokeweed mitogen; TSS-toxic shock syndrome.

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Monoclonal antibodiesagainstmicro-organismsLehner

block proliferative responses of human CD4+ cell lines [4]. As the current candidate vaccine gp160 contains the c&oxy-terminal pentamer which may elicit antibodies to cla.ss II antigens, it seems advisable to remove the pentamer residue from gp160. This study emphasizes the importance of establishing the amino acid sequences of my potential vaccine, as it may point to cross-reactive determinants with self antigens, which CNI be removed, thereby preventing a potential autoimmune reaction. mAb to the core protein ~15, p24 and ~18 were used initially for viral protein analysis, detection of antigenic expression and diagnosis in infected cells (Veronese-Dimatzo et aL, Proc NatlAcudSci US4 1985,82:519+5202; Chassagne et al, J Zmmunol 1986, 136: 144221445). These mAb have now been applied to diagnosis of HIV in 121 lymph node biopsies from patients with general ized lymphadenopathy and AIDS [ 51. There is a spectrum of histological changes in the lymph node architecture. A massive follicular hyperplasia at one end, always manifesting HlV core protein in the germinal centre and lymphocyte depletion at the other end of the spectrum, with loss of follicles and CD4 cells and no detectable HlV core protein. mAb to Pneumocystis curinii are helpful in the diagnosis of pneumonia. Lung infection with P. curinii is a common manifestation in AIDS and the diagnosis can be fraught with difficulties in trying to differentiate yeasts from P curinii by fight microscopy. A combination of three mAb to P. carinii applied by the imrnunofluores-

cence technique offers a specific, simple, fast and sensitive method for the diagnosis of P. curinii, both in bronchoalveolar lavage and lung biopsy specimens [6]. mAb to many other micro-organisms involved in AIDS are readily available [e.g. herpes simplex virus (HEW)].

Differential sensitivity of detection of micro-organisms by mAb as compared with DNA hybridization The possibility that detection of a micro-organism by monoclonal antibodies might be less sensitive and/or specific than the more recent application of DNA (or RNA) hybridization has to be carefuliy examined. A comparison was made between conventional isolation of HSV, immunofluorescence with an fluorescein isothiocyanate (FITC)-labelled, mAb to HSV and DNA hybridization of three cloned fragments of HEW-1 and labelled with biotin [7]. They tested 504 specimens; out of which HSV was positive in 105 specimens; 89% of these were detected by a cytopathic effect in cell culture, 97% by immunofluorescence using mAb and 98% by DNA hybridization. This study suggests that the sensitivity and specificity of the two methods are comparable though further attention will have to be paid to clinical specimens and the convenience of using simple diagnostic kits, without the requirement of specialized microscopical exper-

Fig.1. Passive immunization with mon-

Micro-organism

oclonal antibodies (mAb): (a) Central: by neutralization of the toxin or binding the antigen by mAb which binds to Fc receptors on macrophages or eosinophils in the antibody dependent cytotoxicity reaction. (b) Peripheral: mAb bind the micro-organism, which is prevented from adhering directly to the mucous membrane and may be shed in the mucus (or saliva) or removed by neutrophils passing through the mucosa.

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Immunity to infection

Prevention

of microbial

passive immunization

infection

by systemic

with mAb

Targetting mAb to speci6c microbial epitopes concerned with toxicity or adherence to epithelial surfaces has generated a great deal of interest in passive immunization with mAb

Preventionof Staphy/ococcus aureus-induced toxic shock syndrome by passivesystemicadministrationof mAb The toxic shock syndrome (TSS)

is an acute disease caused by a toxin, originally termed enterotoxin which is secreted by S. aureuS The TSS toxin may act in a+ssociation with endotoxin derived from Gram-negative bacteria. Some mouse n-&r against TSS toxin were able to inhibit TSS toxin-induced rnitogenesis and the induction of interleukin 1 (IL-l) production by human monocytes [8]. In vim experiments to test the ability to protect rabbits from the efffect of infusion of TSS toxin showed that whilst six out of seven control rabbits died, only one of seven rnAb-treated rabbits died. The m&treated rabbits had an attenuated clinical illness and were protected from renal and hepatic failure.

Preventionof infection with mAb against lipopolysaccharides X-linked immunodelicient

mice are unable to produce antibodies to the O-specific carbohydrate determinants of lipopolysaccharide (LPS) and are therefore very sensitive to infection with Pseuabmonus aeruginozu. In a curious reversal of the conventional approach using mouse mAb, human IgM mAb were prepared to the carbohydrate portion of the LPS of P. aeruginw and administered intravenously to mice [ 93. The protective efficacy of passive immunization was assessed by testing the I.Dsu af ter challenge with P. aeruginosa. Administration of a control mAb resulted in an LDs0 of 1.3 x 102, as compared with the spectic mAb which raised the IDso to 6.1 x 104 units. It appears that male X-linked mice which are unable to produce antibodies to LPS can be protected from infection by J? aeruginasa by passive immunization with a human mAb to LPS from this organism. Protection with mAb from Pseudomonas skin infection was also demonstrated in burnt mice and from peritoneal infection in leukopenic mice [lo]. Lethal challenge of CD1 mice with Escbetiia coli also showed protection by passive immunization with mAb speciEctoE. coliLPS [11].AnIgG3antibodytoI.PSwas as effective as an IgG2a antibody to LPS, although the latter was more efficient in binding C3. It seems that the isotype is not a determining factor in protection, as in a murine brucellosis model passive immunization with IgGl, IgG2a or IgG3 mAb to IPS-A from Brucella abor04s equally reduced colonization with this organism [ 12].

Protectionof infection with IgE classof mAb against Schistosomamansoni

IgE class of mAb are not raised by conventional immunization. However, first immunizing with S. munxmi, followed by a glycoprotein-enriched fraction of worms, and then fusion of the mesenteric lymph node cells with myeloma cells resulted in IgE n-&b [ 131. The function of this mAb in vitro was to kill .S, munsoni schistosomula in the presence of eosinophils or macrophages, by the antibody-dependent cellular cytotoxicity mechanism. In vivo passive transfer of IgE mAb revealed significant pro tection against infection when challenged by S. munsoni.

Prevention

of microbial

colonization

by local

passive immunization IgG class of mAb to Streptococcus mutuns cell surface antigens were prepared and applied directly to the teeth of non-human primates. This prevented colonization by S. mutuns and the development of dental caries (Lehner et al, Infect Immun 1985, 50:796-799). Local passive irrmunization was then attempted in humans, which also showed signilicant and long-term reduction in coloniza tion by S. mutans [14]. The isotype of mAb was again not a determinin g factor, as both IgGl and IgG2a antibodies were protective, but epitope specificity appeared to be most important in preventing colonization of strep tococci (Ma et al, personal communication). An important finding in the application of mAb to the prevention of bacterial colonization was the unexpected long duration of protection of over 1 year, when the functional mAb is unlikely to have remained in situ longer than l-2 weeks. This might be interpreted by a shift in bacterial ecology, for the place vacated by S. mutans is filled by another organism from the oral flora, thereby preventing colonization by S. mutuns Local passive immunization of other mucosal surfaces with a mixed microbial flora might be an extension of this type of passive immunotherapy. IgA class of mAb are obvious candidates for mucosal protection, but IgA mAb are not produced by the conventional immunization procedures. However, mucosal immunization elicits IgA class of mAb and these were raised to Sendai virus [ 151. The mAb showed in vitro neutralization activity. Passive intranasal administration to the respiratoiy tract of mice in vivo resulted in complete pro tection from infection and sign&ant reduction in virus titres, when compared with control mice immuni7ed with an unrelated mAb of IgA class. In conclusion IgG and IgM classes of mAb can prevent central-vascular infection by S. aureus and LPS-producing Gram-negative organisms. IgG and IgA classes of mAb were capable of preventing mucosal colonization of bacteria and IgA mAb of viruses, whereas the IgE class of mAb was protective against parasitic infection. This illustrates the broad spectrum of functional applications

Monoclonal

of mAb, with some differential activity of each isotype, though this may not be a general rule.

Humanized

and human mAb

The prophylactic and therapeutic application of mAb in human subjects makes it desirable to use mAb with minimal chances of eliciting an immune response. Rodent mAb Induce antibodies, though this is unlikely to be a problem ln passive mucosal immunization. In this very brief survey no attempt will be made to review the field, particularly as a comprehensive survey has been carried out recently [ 161. Furthermore, a review of human mAb produced by in vitro immunization has just been published [ 171. Instead, the interested reader will be directed to these reviews and only a ritsume of the state of the art in humanized and human mAb will be attempted. Humanized

mAb

Human

mAb

Great efforts have been made to produce human mAb, with variable success, as early as 1977, by Stein& et al. The source of Immune lym(Nature 1977,269:420-422). phocytes is restricted to peripheral blood lymphocytes, though tonsils and spleens are also available. The state of immunity of these cells is dependent on the history of immunization of the individual but art&al immunization can be carried out with the conventional microbial vaccines in order to boost immunity. This hurdle appears to have been overcome by in vitro immunization of peripheral blood lymphocyte after purging the cells with the lysosomotropic methyl ester of leucine (Leu-OMe). The latter eliminates killer cells and suppressor cells and leaves enriched B cells, CD4+ T cells and accessory cells (dendritic cells) which respond specifically to antigen in vitro [ 171. In vitro immunization has to be supported by IL-2, interferon y and B cell factors derived from pokeWeed mitogen (PWM)-stimulated human T cells that were irradiated. The primed cells were then fused with a human lymphoblastoid cell line or a mouse plasma cytoma cell line, resulting in human-human or human-mouse hybrids which secrete human Ig. Human peripheral blood lymphocytes were used successfully by the Leu-OMe method to prepare human

against

micro-organisms

Lehner

mAb to recombinant

gp120 of HIV [ 171. These human hybridomas maintain a stable phenotype in culture for periods of more than 5 months. Human mAb against varicella-zoster virus were successfully produced by in vitro stimulation with viral antigen and PWM, without using ku-OMe, and the cells were fused with a mouse myeloma [18]. It seems that human mAb can now be produced with increased confidence by in vitro stimulation of peripheral blood lymphocytes, but the most appropriate fusion partner giving long-term stability for the antibody secreting hybridoma needs to be determined.

Annotated

references

and recommended

reading 0 ??e

One approach to overcome the foreign nature of rodent mAb is to humanize it. This is a hybrid molecule consisting of mouse variable and human constant region domains (Jones et al, Nature 1986, 321522-525; Sahagan et al, J ImmunoZ;l986, 137:106&1074) reviewed by Oi and Morrison (Biotechiques 1986, 4:214). The advantage of this human-mouse hybrid is that the mAb retain the specificity of the mouse mAb but they will be less immunogenic because the constant portions of the molecule which constitutes most of the mAb are human. To humanize mAb requires specialized genetic recombinant technology, not commonly available in most laboratories.

antibodies

1. 00

Of interest Of outstanding interest KRUSTB, LAURENTAG, LE GUERN A, JEZANNEQUIN 0, MONTAGNIER L, HOVANESS~AN AG: Characterization of a monoclond an-

tibody spectic for the HIV-precursor glycoprotein. AIDS

1988, 2:17-24. Describes an mAb to gp160 of HlVl which is a candidate vaccine against AIDS,as most seropositivesubjects produce anti-gpl60 antibodies. 2. ??

PIATA F, A~TRAN B, MARTINSLP, WAINHOBSON S, RAPHAELM, MAYAUD C, DENE M, GUILLON J-M, DEB& P: (Let&r to the

Editor) AIDS-virus-specific cytotoxic T lymphocytes in lung disorders. Nature 1987, 328:348-350. m.kb to gp160 used to detect and purify gp160 from transfected cells. THOMASEK, WEBER JN, MCCLIJREJ, CLWHAM P, SINGHAL M, SHRNER MK, WEISS RA: Neutralizing monoclonal antibodies to the AIDS virus. AIDS 1988, 2:25-30. mAb to gpll0 were mised which neutralize HIV-1 by inhibition of infection and these as well as anti&i1 inhibit syncytial formation. These are important analytical reagents recognizing a neutralization epitope.

3. a.

4. ??e

GILDINGH, ROBS%FA, GATES FT III, IINDER W, BEINING PR, HOFFMAN T, GUIDING B: Identification of homologous re-

gions in human immunodeficiency virus I gp41 and human MHC class II p 1 domain I. Monoclonal antibodies against gp4lderived peptide and patients’ sera react with native HIA class II antigens, suggesting a role for autoimmunity in the pathogenesis of acquired immune deficiency syndrome. J EqO Med 1988, 1673914-923. This paper raises an important issue of cross-reactiviry between a 5residue peptide in gp41 of HIV-1 and HIAclass II antigen. mAb against this common epitope cross-react both with the native HIV-1 and class II antigen. Serum anti-class II antibodies were found in V3 of AIDS patients and these antibodies may contribute to an autoimmune process in AIDS. 5. ??

TENNER-R&Z K, F&z P, DETRICH M, KERN P, JANOSSVG, VERONESE-DIMARZO F, KLA’~IANN D, GLUCKMANJ-C, Po~ovlC M: Monoclonal antibodies to human immunodeficency virus:

their relation to the patterns of lymph node changes in persistent generalized lymphadenopathy and AIDS. AIDS 1987, 1:95-104. An application of mAb to the core proteins to detect HIV-1 in relation to a spectrum of lymph node changes in AIDS. 6.

GJLLVJ, EVANSG, SToCK F, PARIULI~ JE, MAXJRH, KOVACSJA: carinii by fluorescent-antibody stain using a combination of three monoclonal ant&dies. J Clin Microbial 1987, 25:1837-l&10. mAb to P. cufinii is useful in the diagnosis of this lung infection, commonly presenting in AIDSpatients. ??

Detection of PneumaysCis

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Immunity to infection 7. 0

ESPY MJ, Swnr TF: Detection of herpes simplex virus in conventional tube cell cuhures and in shell vials with a DNA probe kit and monoclonal antibodies. J Clfn Mfcrobfol 1988, 26:22-24. This investigation compares the sensitivity and specificity of detection of HSV between immunofluorescence by using mAb and DNA hybrkiization. BONVENIXE PF, TrIOMF’sON MR, ADINOLFl LE, GIUIS ZA, PARWNN!ZTJ: Neutrahxation of toxic shock syndrome toxin1 by monoclonal antibodies in vftro and in vfvo. Infect Immun 1988, 56:135-141. A potentially important therapeutic application of mAb in preventing in rabbits the toxic shock syndrome induced by a .S. anzeuS enterotoxin. 8.

??

9. 0

Z~EERINKHJ, GAMMONMC, HUTCHLWNCF, JACKWN JJ, PIER GB, PUCKETTJM, SEWELLTJ, SIGALNH: X-Linked immunodeficient mice as a model for testing the protective efficacy of monoclonal antibodies against Pseudomonus aerugf~osu. Infect Immun 1988, 56:120+1214. A human mAb against the LPS of P. aeruginosa was administered to mice and this protected them from lethal infection with this o&m. 10. e.

SAWADA S, XAWAMLIRA T, hl.w~~o Y: Immunoprotective human monoclonal antibodies against five major serotypcs of Pseudomonas aerugfnosa. J Gen Microbial 1987, 1333581-3590. Human mAb to the 0 antigen of P. uerugimxa WS were prepared by fusing tonsillar lymphocytes with mouse myeloma cells. Passive transfer of these mAb protected mice from Pseudomonas skin infection in burnt mice. 11. 0

COIJGHLIN ET, BOGARD EC JR: Immunoprotective murhre monoclonal antibodies specific for the outer-core poiysaccharide and for the O-antigen of Escberfcbfu coli 011 l:B4 lipopolysaccharide (LPS). J Immunoll987, 139:557-561. Passive immunization with mAb to LPS from E. cdiprotected mice from lethal infection with E co/i 12.

LLMETJ, PLOMMETAM, DUEMY G, PIQMMET M: Immunity conferred upon mice by anti-WS monoclonal antibodies in murine brucellosis. Ann Inst Pasteur Immunol 1987, 138:417-424. mAb against the LPS-A of Brucelh abortus reduced infection with this organism, irrespective of the IgG subclass, ??

VERWAERDE C, JOSEPH M, CAPRONM, PIERCERJ, DAMONNEWLIE M, VELGE F, AURLWLTC, CAPRONA Functional properties of a rat monoclonal IgE antibody specitic for Schistosoma mansoni. J Immunol 1987, 13844414446. A technique of raising IgE class mAb against .S. munsoni that kills in vitro schistosomula, in the presence of eosinophils or macrophages. In viva administration of the mob showed significant protection against infection by S. munsoni 13.

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14. ee

MAJR-C, SMI’IWR, IEHNERT: Use of monoclonat antibodies in local passive immuniaation to prevent colonization of human teeth by Streptococcus mutans. Infect Immun 1987, 55:1274-1278. This investigation shows that application of mAb in mucosaf passive immunization in human subjects prevents colonization with Strqxoux cus mutans The long-term effect of mAb preventing specific bacterial colonization in a mixed microbial environment makes this a significant prophylactic approach in mucosal immunity. M%?ANEC MB, NEDRUDJG, LWM ME: Immunoglobulin A monoclonal antibodies protect against Sendai virus. J Viral 1987, 61:262&2626. A technique of producing IgA class of mAb against Sendai virus. Passive inn-a-nasal administration of rn.Ab protected mice from respiratory tract infection with the virus. 15.

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JAMESK, BELL GT: Human monoclonal antibody production - current status and future prospects. J Immunol Methods 1987, lOCk5-40. A comprehensive review of the production and application of human mAb. 16.

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BORREBAECK CAX, DANIEISSONL, MWER SA Human monoclonal antibodies produced by primary in vitro immunixation of peripheral blood lymphocytes. Proc Nut1 Acud Scf us4 1988, 85:3995-3999. A signliicant advance in raising human mAb is described by in vitro stimulation of peripheral blood lymphocytes, alter me cells are treated with the lysosomotropic a-leucine methyl ester, 17. ??e

18.

SUGANOT, MATWMOTOY, MNAMOTOC, MAWHO y: Hybridomas producing human monoclonal antibodies against varicella-zoster virus. Eur J Immunol 1987, 17:35‘+364. Human mAb against varlcella-zoster virus by in uitro stimulation of pe ripheral blood lymphocytes with me viral antigen and PWM. ??