A Combinatorial Phage Display Library for the Generation of Specific Fab Fragments Recognizing Human Spermatozoa and Inhibiting Fertilizing Capacity In Vitro¹

^a Department of Molecular Biology and Biotechnology ^b and Obstetrics and Gynaecology, University of Sheffield,Sheffield S10 2UH, United Kingdom

	ABSTRACT

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To select a source of lymphocytes for the generation of an anti-sperm-biased combinatorial phage display library, venous blood was obtained from 34 vasovasostomy (vasectomy reversal) patients approximately 3 mo after surgery. Using a variety of immunoassays, serum was analyzed for antibodies against human spermatozoa, and a patient was selected on the basis of high titer of antibodies that recognized the equatorial region of the sperm head and inhibited sperm fertilizing capacity in vitro. Total RNA isolated from the stored lymphocytes of this individual was reversed transcribed, and 1 (Fd) region and chains were amplified by polymerase chain reaction for the successful construction of an antibody phage display library. The library was panned against human spermatozoa to isolate sperm-specific phage that recognized the equatorial region of the sperm head. Three preparations of Fab were tested via the hamster egg penetration test. Each preparation significantly (p < 0.005) inhibited sperm-egg binding and fusion, with one preparation (designated Fab-G) causing complete inhibition. Sequence analysis of the light gene encoding Fab-G revealed a 93% homology with the light chain of human anti-human immunodeficiency virus gp120 p35 variable region. This technology may have a practical application in characterization of the immune response to spermatozoa and for the design of sperm-based contraceptive vaccines.

	INTRODUCTION

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Although still at an experimental stage, immunocontraception is emerging as a novel alternative to conventional contraceptive methods [1]. Usually an active immunization protocol is envisaged with vaccines evoking antibodies that inhibit a key component of the reproductive system such as chorionic gonadotropin [2], zona pellucida protein [3, 4], or sperm antigens [5–9]. This method offers the possibility of relatively long-term contraception that can be tissue or cell specific. At present, however, there are several major drawbacks. Our knowledge surrounding the reversibility of these vaccines is limited, and there is a possibility of inducing immune-complex diseases and nonspecific cell-mediated autoimmunity [10]. Secondly, there may be a qualitative and quantitative variation in the immune response generated that might alter the efficacy of the vaccine [11]. Finally, there is a variable lag period of several weeks after immunization before contraception is attained.

Passive immunization avoids the induction of a systemic humoral and cell-mediated response and therefore circumvents many of the problems associated with vaccination. Hence it could provide a controlled short-term approach for testing immunocontraceptive mechanisms, or perhaps it could be used directly as a means of regulating fertility. A major difficulty has been to obtain a purified antibody preparation against a known antigen that might inhibit fertility and that does not itself evoke an immune response (i.e., homologous antibodies). However, in the last few years, the combinatorial antibody phage display technique has been developed to generate specific human monoclonal antibodies [12–15]. This method is achieved by fusing the coding sequence of the antibody variable regions to the amino terminus of phage minor coat protein cpIII. The random fusion of heavy- and light-chain fragments of immunoglobulin results in a diverse antibody library where the phenotypic-genotypic relationship provides a direct link between antibody-binding regions and the genes from which they were encoded. In this way, phage expressing a specific Fab can be cloned so that monoclonal fragments can be generated in the periplasm of bacterial host cells for conventional extraction and purification. Moreover, antibody binding can be altered, if necessary, by modification of the variable regions of the assembled Fab DNA. In theory, a naive combinatorial library (i.e., from an nonimmunized individual) containing approximately 10¹³ antibody variants can be screened against any known antigen to obtain a monoclonal antibody. In practice, it is often better to use an antigen-biased library from a patient who has generated antibodies against the antigen(s) under investigation [16]. This library might be smaller than a naive library, but it should contain a greater proportion of antibody clones relevant to the antigen(s) in question.

Several sperm antigens are now candidates for contraceptive vaccines [2, 5, 7–9, 17]. These sperm proteins were mainly identified initially from studies in laboratory animals, and it remains unclear whether they will be suitable immunogens in a vaccine for human use because the appropriate tests require human volunteers. An alternative approach is to investigate men and women with high titers of antibodies to spermatozoa and attempt to correlate the presence of specific antibodies with infertility [18]. However, autoimmunity to spermatozoa may arise for a number of reasons (e.g., antigen mimicry) and may be unrelated to the onset of infertility. In this respect, men that have undergone a vasovasostomy (vasectomy reversal) represent a well-defined group of patients (of previous proven fertility) who are otherwise healthy but can develop an autoimmune reaction and exhibit high titers of antibodies against spermatozoa associated with persistent infertility, even though the ejaculate contains adequate numbers of spermatozoa [19–21]. In effect, such individuals have been naturally immunized, and thus characterization of their antibodies and corresponding antigens may provide important information for contraceptive vaccine design.

Using peripheral blood lymphocytes from one such vasovasostomy patient, we describe here the generation of human Fab fragments, using phage display technology, that recognize human spermatozoa and inhibit sperm fertilizing capacity in vitro.

	MATERIALS AND METHODS

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Collection and Preparation of Blood Samples

Peripheral venous blood samples were obtained by consent from 34 vasovasostomy patients attending the andrology clinic (Jessop Hospital for Women, Sheffield) approximately 3 mo after surgery. Informed consent was obtained from patients, and the project was approved by the South Yorkshire research ethics committee.

For preparation of serum, blood (5 ml) was drawn to sample tubes without anticoagulant and incubated at room temperature for 1 h. After centrifugation at 500 x g for 10 min, the serum was recovered by aspiration and stored in aliquots (0.5 ml) at -20°C until analyzed in immunoassays. A peripheral venous blood sample was also collected from a seronegative donor. The serum recovered from this individual was included as a negative control in all assay procedures.

To obtain peripheral blood lymphocytes, blood was drawn into heparin-coated tubes and diluted in an equal volume of sterile PBS (120 mM sodium chloride, 2.7 mM potassium chloride, and 10 mM phosphate-buffered salts, pH 7.4). A maximum of 25 ml of diluted blood was overlayered onto 12.5 ml of lymphocyte separation medium (Lymphoprep; ICN Pharmaceuticals, Costa Mesa, CA) and centrifuged at 470 x g for 35 min at room temperature. The lymphocyte layer was recovered by aspiration, diluted in 20 ml of PBS, and centrifuged at 1300 x g for 15 min at 4°C. The cell pellet was stored at -80°C before use.

Immunoassays

Serum samples were analyzed for antibodies against spermatozoa by four assays: an ELISA, based on the method described by Herr et al. [22]; indirect immunofluorescent localization (IIF) [23]; the tray agglutination test (TAT) [24]; and the hamster egg-sperm penetration test (HEPT) [25].

ELISA assay The procedure described by Herr et al. [22] was followed with slight modifications. Aliquots (100 µl) of washed semen samples from various donors were plated at a sperm concentration of 3 x 10⁶/ml in wells of Maxisorp Immunoplates (Nunc, Roskilde, Denmark) and allowed to dry overnight at 37°C. The plate was blocked with 1.5% nonfat milk powder in Tris-buffered saline with Tween 20 (TTBS; 50 mM Tris, 138 mM sodium chloride, 2.7 mM potassium chloride containing 0.05% Tween 20) for 2 h at 37°C. After a washing step, aliquots (100 µl) of serum (patient and seronegative control) at 1:250 dilution were added to wells and incubated for 1 h at 37°C. Following an additional washing step, aliquots (100 µl) of affinity-purified goat anti-human IgA + IgG + IgM (heavy and light chain) conjugated with horseradish peroxidase (ICN Pharmaceuticals) were added at a dilution of 1:1000 to each well, and the plate was incubated at 37°C for a further hour. Colimetry was developed with 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid substrate (Sigma-Aldrich Company, Poole, Dorset, UK). After 15 min, absorbance was measured at a wavelength of 405 nm on a plate reader (Milenia Kinetic Analyzer; Diagnostic Products Corporation, Los Angeles, CA). A range of primary and secondary antibody and antigen controls were included on each plate. Each serum sample was assayed in triplicate.

IIF localization The ability of antibodies in serum samples to bind to methanol-fixed or viable washed human spermatozoa was assessed using methods described previously [5]. Briefly, serum was diluted 1:50 in PBS and incubated with spermatozoa either on a slide or in a microtube for 1 h at 37°C. After three washes in PBS, primary antibody-binding sites were localized by an incubation with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-human IgG (Sigma-Aldrich) at a dilution of 1:50 for 1 h at 37°C. After further washes, sperm samples were mounted [23] and examined by epifluorescent microscopy (Olympus, Tokyo Japan; BH-2 with UV filter, 390-450 nm). Appropriate primary and secondary antibody controls were included for comparison.

TAT Sperm-agglutinating antibodies in serum were detected by the TAT [24] with minor modifications. Aliquots of serum were heat inactivated at 56°C for 30 min and diluted in PBS to produce doubling dilutions (50 µl), ranging from 1:4 to 1:8192, in a 96-well plate. An aliquot (10 µl) of washed sperm suspension (10⁷ sperm/ml) from a donor of proven fertility was added to each well and incubated overnight at room temperature. The degree and type of agglutination were examined using a phase-contrast inverted microscope (Olympus CK-2). The agglutination titer was determined as the reciprocal of the highest dilution at which agglutination was observed to occur. The agglutination patterns were characterized as head-head, tail-tail, or mixed.

HEPT The protocol used was as described previously [25]. Before the addition of zona-free oocytes, heat-inactivated patient or control serum (1:10 dilution) was added to capacitated sperm suspensions. The oocytes were incubated with spermatozoa for 16 h (to give maximum opportunity for sperm penetration) and were examined for the presence of sperm bound to the oolemma, decondensed sperm heads, and attached tails in the cytoplasm [25]. A mean of 30 oocytes was examined for each serum sample. The sperm penetration index (SPI) was calculated by dividing the percentage of oocytes penetrated in the test sample by that in the control sample and multiplying by 100. A value < 50% was considered to display inhibition of penetration.

Isolation of RNA and Construction of Antibody Phage Display Library

Lymphocyte RNA was prepared from the selected patient using an RNA isolation kit (Ultraspec, total RNA isolation reagent; Biotex Laboratories Inc., Houston, TX) according to the manufacturer's instructions. Total RNA (2.5 µg) was reverse transcribed, and 1 (Fd region) and chain cDNA was amplified by polymerase chain reaction as described elsewhere [16] with minor modifications. Briefly, a set of predesigned heavy- and light-chain primers was utilized to amplify the different families of the 1 heavy and light chains. The primers were designed to incorporate specific restriction sites that would be unique for ligation of the antibody DNA into the pComb3HSS phagemid vector (gift from Scripps Institute, La Jolla, CA). Polymerase chain reaction products were separated on a 2% agarose gel, and the bands at 660 base pairs were excised and purified using a commercial purification method (Wizard; Promega Ltd., Southampton, UK). The resulting light chain was digested in a double digest with an excess of the restriction enzymes SacI and XbaI (Promega Ltd.). Typically 80 ng of the digested light chain was ligated with 224 ng of SacI/XbaI-linearized pComb3HSS vector (isolated by agarose gel electrophoresis) in a total volume of 20 µl with 1 unit of T4 DNA ligase (Gibco BRL Ltd., Paisley, UK) at 17°C overnight. To determine the insert frequency of this ligation, phagemid DNA was recovered according to the method of Burton et al. [16]. Ten colonies were selected, digested with SacI and XbaI, and purified by agarose gel electrophoresis. A band of 660 base pairs was indicative of a cloned insert.

The resulting heavy chain was sequentially digested with an excess of the restriction enzymes SpeI and XhoI (Gibco BRL Ltd.). Typically 1 µg of the digested heavy chain was ligated with 6 µg of the SpeI/XhoI-linearized light chain-containing phagemid DNA, and the heavy- and light-chain insert frequency was determined as described above. Nucleic acid sequencing was carried out on recovered double-stranded DNA to determine the sequences of the heavy- and light-chain inserts of a selected clone. The sequences were compared with existing sequences stored in the database by the BLASTN program (National Center for Biotechnology Information, Bethesda, MD), and the derived amino acid sequences were determined by the ALIGN program (Scientific and Educational Software, Durham, NC). The phagemid DNA was then rescued by helper phage, VCS-M13 (Stratagene Ltd., Cambridge, UK), to generate a combinatorial Fab library expressed on the surface of filamentous phage, according to the method described by Burton et al. [16]. The prepared phage were screened immediately by the panning procedure to avoid proteolysis of the Fab fragment from the phage surface.

Panning of the Phage Display Library against Human Spermatozoa to Isolate Sperm-Specific Phage-Fabs

The panning protocol was essentially similar to that described elsewhere [12] but with modifications for human spermatozoa. ELISA plates were prepared as described above, but spermatozoa were fixed with 100% methanol for 30 min. The wells were rinsed twice with distilled water, blocked with 3% BSA (Sigma-Aldrich) in Tris-buffered saline for 1 h, and then incubated with phage suspension (10¹² pfu/ml) for 2 h at 37°C at 100% humidity. Controls included wells with no sperm target and incubation of phage with no cloned inserts. The plates were then washed ten times in TTBS with vigorous pipetting to remove nonspecific phage. Sperm-specific phage were eluted from each well with 1% BSA in 0.1 M HCl, pH 2.2 (adjusted with glycine) and pooled, and the solution was neutralized with 2 M Tris buffer, pH 8.0. The phage were propagated by infecting Escherichia coli XL1-Blue cells (Stratagene Ltd.) for overnight incubation, followed by their recovery and three more rounds of panning. At each round of panning, the immunolocalization of phage on washed donor spermatozoa was assessed by IIF using a goat anti-Fd bacteriophage-FITC-conjugated antibody (Sigma-Aldrich). Finally phagemid DNA was isolated using a midiprep purification procedure (Qiagen plasmid midikit; Qiagen, Dorking, UK). The frequency of inserts was determined as already described.

Preparation of Soluble Fab Fragments

Soluble Fabs were generated by NheI/SpeI (Gibco BRL Ltd.) excision of the gIII fragment. The self-ligated phagemid lacking the gIII fragment was transformed into XL1-Blue cells. Seven selected clones were grown in 10 ml of Superbroth (30 g bacto-tryptone, 20 g bacto-yeast extract, 10 g 3-[N-morpholino]propanesulfonic acid) containing 20 mM MgCl₂ and 50 µg/ml of carbenicillin (Sigma-Aldrich) for 6 h at 37°C. The cells were induced with 1 mM isopropyl-ß-D-thiogalactopyranoside (Sigma-Aldrich), incubated overnight at 30°C, centrifuged at 1500 x g for 15 min, and resuspended in 1 ml of PBS. Soluble Fabs were extracted by freezing the cell suspension in a dry ice-ethanol bath for 5 min followed by thawing at 37°C. This freeze-thaw process was repeated four times before the cell debris was pelleted by centrifugation at 8000 x g for 5 min and the supernatant containing the Fabs recovered by aspiration.

Characterization of Sperm-Specific Fab

Immunoblotting analysis of soluble Fab was carried out according to methods described elsewhere [26–32]. After excision of the gIII fragment, the light chain was recovered from a positive clone and sequenced as described above. Binding of Fabs to spermatozoa was determined by IIF, and their effect on sperm fertilizing capacity was assessed using the HEPT.

	RESULTS

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Characterization of Serum and Selection of Vasovasostomized Patient

Of 34 patients examined over a 12-mo period, 23 (68%) exhibited sperm antibodies in their serum as detected by ELISA. A sample was considered seropositive when absorbance values were equal to or exceeded twice background. In the TAT, 26 serum samples (76.5%) exhibited a titer above 1:64, and 50% of these samples had titers of 512 or above. When the type of agglutination was characterized, 47% of samples evoked head-head agglutination, 29% tail-tail agglutination, and 24% mixed agglutination. The latter pattern was correlated with low agglutination titers. By IIF, 23 samples (68%) displayed antibody binding on methanol-fixed spermatozoa. In 26.5% of cases immunolocalization was to the entire acrosome (apical and equatorial region), and in 17.6% of cases it was to the equatorial region (Fig. 1). Immunofluorescent localization was detected on 68% of viable spermatozoa with characteristic "capping" of antigen. In all, 11 serum samples displayed positive titers in the ELISA and TAT and showed IIF localization with methanol-fixed and viable spermatozoa. Four of these serum samples were tested for their ability to inhibit human sperm penetration of zona-free hamster eggs in vitro, and two were shown to significantly (p < 0.005) inhibit sperm-egg binding and fusion (Table 1). The patient (21VV) who was the donor of one of these samples was therefore selected as the source of lymphocytes for construction of the library. The serum was strongly seropositive in ELISA, displayed a TAT titer of 1:4096 (with a characteristic agglutination of sperm heads at the equatorial region), showed immunolocalization to the equatorial region of the sperm head, and inhibited sperm-egg fusion (SPI 13.9%).

View larger version (95K): [in this window] [in a new window]   FIG. 1. IIF localization of antibodies on human methanol-fixed donor spermatozoa using serum recovered from patient 21VV. FITC staining over the equatorial region. Bar = 2 µm.

Library Construction

The number of individual clones generated by the transformation indicated the number of library members. Background levels were determined by transforming the equivalent amount of vector DNA that had been subjected to the ligation process in the absence of heavy- and light-chain inserts. A light-chain library of 3.3 x 10⁷ members was generated. Sequential cloning of the heavy chain generated a complete library (heavy and light chain) of 1.3 x 10⁷ members. Confirmation of correct construction of the library was obtained by sequencing the light- and heavy-chain inserts, which revealed 98% homology with a published sequence as assessed by the BLASTN and ALIGN software programs.

Panning of the Library

The library was panned four times against human spermatozoa. The number of clones generated after each round of panning determined the number of library members (Table 2). There was a small rise in the number of eluted phage over the four rounds of panning. The immunofluorescent staining pattern of phage binding to spermatozoa changed significantly, from no staining detected on the first round of panning to staining at the edges of the equatorial region after round 3 (Fig. 2a) and distinct staining over the equatorial region on the final round of panning (Fig. 2b). This binding pattern was the same as the corresponding antibody-binding pattern of the serum for this individual (Fig. 1).

View larger version (102K): [in this window] [in a new window]   FIG. 2. a) Immunofluorescent localization of the phage-Fabs recovered from round 3 of panning against human methanol-fixed donor spermatozoa. Bar = 1.5 µm. b) Immunofluorescent localization of the phage-Fabs recovered from round 4 of panning against human methanol-fixed donor spermatozoa. Bar = 1.5 µm.

Characterization of Soluble Fab Fragments

SDS-PAGE and immunoblotting revealed the presence of both heavy and light chains of the purified soluble Fab from different clones. Of seven preparations of Fab produced from different clones, five recognized the equatorial segment on human spermatozoa when tested by IIF (Fig. 3).

View larger version (72K): [in this window] [in a new window]   FIG. 3. IIF localization of Fab fragments on human methanol-fixed donor spermatozoa using recombinant soluble Fabs recovered from clone G. FITC staining over the equatorial region. Bar = 4 µm.

Effect of Fab Preparation on Sperm Penetration of Zona-Free Hamster Oocytes

The three preparations of soluble Fabs displaying the strongest affinity for the equatorial region of human spermatozoa as detected by IIF were assessed for their effect on the capacity of human spermatozoa to fuse with zona-free hamster oocytes in vitro as described above. A control (Fab-A) was included that contained heavy and light chains but did not recognize sperm. Soluble Fab at a final concentration of ~10 µg/ml was added to capacitated sperm suspension 30 min before the addition of oocytes. Each preparation significantly inhibited sperm-egg binding and fusion, with one preparation (designated Fab-G) completely inhibiting sperm-egg interaction without interference of sperm motility (Table 3).

Sequence Analysis of the Light Chain for Clone G

The gIII fragment was excised from the clone responsible for Fab-G (inducing strong inhibition of sperm-egg fusion), and the light chain was sequenced. Nucleotide sequencing data revealed a 93% homology between Fab clone G light-chain sequence and the human anti-human immunodeficiency virus (HIV) gp120 p35 light-chain variable region sequence (Fig. 4, a and b).

View larger version (28K): [in this window] [in a new window]   FIG. 4. a) The derived nucleotide sequence of the isolated light-chain insert determined from DNA prepared from clone G. The complimentary determining regions (CDR) are indicated in bold print. b) The corresponding amino acid sequence of the Fab-G light-chain insert (CLONE). Homology to the human anti-HIV gp120 antibody p35 light-chain variable region sequence is also indicated (SUBJECT). The CDR regions are indicated in bold print.

	DISCUSSION

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Although the combinatorial library method has been used to generate human antibodies without immunization, there remains considerable interest in cloning antibodies from immune individuals for prophylaxis, therapy, and the study of the human humoral response. The main objective of the study presented here was to investigate antibody phage display technology for the production of human Fab fragments that recognize human sperm antigens that might be involved in fertilization processes and might be used for contraceptive vaccines. At present, the main approach for identifying such antigens has been to use indirect methods such as assessing whether specific murine monoclonal antibodies inhibit human sperm function [25]. While these procedures have been successful in determining potentially important proteins (conserved across species) involved in fertilization, the link between antigen, immune response, and human infertility has remained tenuous, due in part to the lack of suitable testing in humans. Although at a preliminary stage, the results demonstrate clearly that specific human Fabs can be generated that inhibit sperm fertilizing capacity in vitro. This is a significant finding because the technique now makes it feasible to directly trace a pathway from the induction of antibodies against spermatozoa in an individual to the expression and purification of specific monoclonal antibody species, the genotypes of which can be fully characterized and manipulated, for example, to increase antibody-binding affinity.

Men who had recently undergone a vasovasostomy were investigated because they represent a group of individuals in whom the cause of antibodies to spermatozoa is well defined [19–21] in comparison to other patients in whom the etiology is usually unknown [33]. Moreover, as the onset of an immune response was most likely to occur shortly after surgery, blood samples could be obtained when many activated peripheral lymphocytes would be present (2–3 mo after the operation). Since the measurement of sperm antibodies in serum can be unreliable, several assays were performed. The results displayed reasonable concurrence, with 12 of 34 patients (35%) seropositive in three assays and 11 patients (32%) seropositive in all the assays. Importantly, the sera of three patients with high antibody titers inhibited the ability of donor spermatozoa to penetrate zona-free hamster eggs in vitro, indicating that antibodies that recognized functionally important sperm epitopes were present. In this respect, the serum of the patient whose lymphocytes were used to make the combinatorial library contained antibodies that localized specifically to the equatorial segment of the sperm head. This region is involved in the fusion of the spermatozoon with the oolemma at fertilization [34].

Constructing an antibody library from peripheral blood is considered more difficult than doing so from other immune tissues such as spleen or bone marrow because the latter normally contain significantly more differentiated B cells that spontaneously produce antibodies. However, vasovasostomy patients may represent an exception. The population of differentiated B cells in peripheral blood lymphocytes is increased markedly by recent contact with antigen. It is likely that antibodies are generated after vasovasostomy as a result of leakage of spermatozoa from the vas deferens at surgery. These may evoke new antibodies but also act as an in vivo booster to stimulate specific B cells resulting from an immune response generated during the earlier vasectomy procedure. In this case, the population of peripheral blood lymphocytes obtained for the combinatorial library may more closely represent what might be present after recent immunization and therefore can yield useful information for the development of contraceptive vaccine strategy.

Panning the library against human methanol-fixed donor spermatozoa led to the isolation of phage that specifically recognized the equatorial region of the spermatozoa head. This was not immediately evident on the first round of panning, but with every round thereafter a more intense immunofluorescent staining was detected using specific antibody against phage coat protein (Fig. 2, a and b). This localization was the same as that obtained with the donor serum and may indicate that the combinatorial library is particularly rich in members encoding Fabs for this region on spermatozoa. Certainly, phage selection was confirmed after the final panning when Fab preparations were made from seven clones, five of which bound specifically to the equatorial region. That these antibody fragments might recognize functionally important epitopes was demonstrated using zona-free hamster oocytes. Many previous investigations have shown that human sperm penetration of zona-free hamster oocytes provides a good indication of sperm fertilizing capacity in vitro and in vivo [35, 36]. Since Fabs are univalent they do not normally cause agglutination of spermatozoa, and therefore their capacity to inhibit sperm-egg fusion strongly suggests that the corresponding epitope may play a role in this process, which is known to be initiated at the equatorial region of the sperm head [34]. From an immunocontraceptive perspective it would be interesting to identify any constructed Fabs that interfere with sperm-zona interactions.

The light chain encoding the Fab-G, which was the most effective in blocking sperm-egg fusion, displayed close homology (> 93%) with a sequence in the BLASTN database coding for a light chain that recognizes HIV gp120 protein [37]. At present, the relevance of this finding is unclear. Because many sequences in the database relate to antibodies of HIV, there is likely to be a bias in favor of such a sequence. However, the homology that was found for the light-chain sequence of the Fab-G was considerably higher than for sequences of the other clones, suggesting that the similarity represented a true homology. Since regions of the light chain are involved in recognition of the epitope, this finding may indicate that the sperm-specific Fab has a binding affinity similar to that of the Fab for gp120. In this respect, it is of interest that gp120 is part of the gp41/120 viral coat protein complex that initiates HIV fusion with CD⁴⁺ lymphocytes. Similar fusion proteins may occur in the equatorial region of the sperm head to initiate fusion with the oolemma.

	ACKNOWLEDGMENTS

 
We thank the staff of the Infertility Clinic, Jessop Hospital for Women, for their assistance in collection of blood samples. We are also very grateful to Mr. Miles Fox for his help in this project.

	FOOTNOTES

 
¹ R.C. was supported by a studentship from the British Fertility Society.

² Correspondence. FAX: 0114 272 8697; h.d.moore{at}sheffield.ac.uk

Accepted: July 7, 1998.

Received: May 6, 1998.

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