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Detection and Typing of Herpes Simplex Viruses by Using Recombinant Immunoglobulin Fragments Produced in Bacteria† PAOLA CATTANI,1* GIAN MARIA ROSSOLINI,2* STEFANIA CRESTI,2 ROSARIA SANTANGELO,1 DENNIS R. BURTON,3,4 R. ANTHONY WILLIAMSON,4 PIETRO P. SANNA,5 1 AND GIOVANNI FADDA Istituto di Microbiologia, Universita ` Cattolica del Sacro Cuore, 00168 Rome,1 and Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Universita ` di Siena, 53100 Siena,2 Italy, and 3 Departments of Molecular Biology, Immunology,4 and Neuropharmacology,5 Scripps Research Institute, La Jolla, California 92037 Received 29 October 1996/Returned for modification 3 December 1996/Accepted 6 March 1997
Thirty-seven bacterial clones producing human recombinant monoclonal antibody Fab fragments (rFabs) reactive to herpes simplex virus (HSV) antigens were selected from a human combinatorial antibody library constructed in a phage-display vector by a panning procedure against an HSV lysate. Thirty-four of the HSVspecific rFabs were able to specifically recognize HSV-infected cells in indirect immunofluorescence (IF) assays; of these, 25 recognized cells infected by either HSV type 1 (HSV-1) or HSV-2, while 9 recognized only HSV-1-infected cells. One HSV type-common rFab (rFab H37) and one HSV-1-specific rFab (rFab H85) were further evaluated as reagents for viral detection and typing by IF staining in 134 HSV-positive (72 HSV-1 and 62 HSV-2) viral cultures from clinical specimens. The results obtained with these two rFabs were fully consistent with those obtained with a commercial preparation of fluorescein-labeled anti-HSV type-specific murine monoclonal antibodies. The detection sensitivity with the type-common rFab in indirect IF assays was higher overall than that provided by the type-specific murine monoclonal antibodies. Preparations of rFabs suitable for IF staining can be easily and inexpensively obtained in a clinical microbiology laboratory from Escherichia coli cultures. Similar HSV-specific rFabs, therefore, could be advantageous for in vitro diagnostic purposes. common procedures adopted in a clinical virology laboratory (1, 34). For this purpose, fluorescein-labeled type-common and type-specific monoclonal antibodies have been produced, and although they are more expensive than polyclonal antisera, they yield better results and are currently preferred for HSV identification and typing (3, 14–17, 19, 20, 29, 36). In this work with a panel of HSV-specific human recombinant monoclonal antibody Fab fragments (rFabs) produced in bacteria, including both type-common and type 1-specific clones, we show that similar reagents can be useful for laboratory diagnosis of HSV by indirect IF staining procedure.
Herpes simplex virus (HSV) infections are among the most prevalent viral infections in humans; they are usually responsible for benign recurrent mucocutaneous lesions. Two serotypes of HSV are distinguished. HSV type 1 (HSV-1) is usually associated with mouth and eye infections, while HSV-2 is usually the cause of genital infections acquired by sexual transmission. HSVs are also the most common cause of sporadic viral encephalitis. Moreover, serious or life-threatening HSV infections can occur in newborns after perinatal infection and in severely immunocompromised hosts, including AIDS patients, in which they are a leading cause of morbidity and mortality (2, 31–34). Laboratory diagnosis of HSV infections is crucial to confirm the need for antiviral chemotherapy, while viral typing, although not relevant for therapeutic purposes, is important for epidemiological and prognostic purposes (1, 10–13, 18, 23, 24, 28, 34). Laboratory diagnosis is mainly based on viral isolation and/or direct detection of viral components in clinical specimens (1). Viral culture associated with rapid identification of isolates by immunofluorescent (IF) staining is among the most
MATERIALS AND METHODS Selection of bacterial clones producing HSV-specific rFabs. Bacterial clones producing HSV-specific rFabs were isolated from a human immunoglobulin G1 (IgG1) combinatorial phage-display library constructed from an HSV-1-seropositive healthy donor essentially as previously described (4, 35). Briefly, total RNA was extracted from bone marrow mononuclear cells of the donor and retrotranscribed to cDNA by using commercial kits (RNA isolation kit [Stratagene Inc., La Jolla, Calif.] and first-strand cDNA synthesis kit [Boehringer, Mannheim, Germany]). cDNA was used as the template for PCR amplification of sequences encoding the VH-CH1 region of IgG1 heavy chains (HC) and the Vk-Ck region of k light chains (LC) by using a set of primers previously described (22) and a high-fidelity thermostable DNA polymerase (Pfu; Stratagene). The LC- and HC-corresponding amplimers were sequentially cloned into the pCombIII phagemid vector (4). The repertoires of cDNAs encoding the VL-CL and VHCH1 immunoglobulin domains of the donor (amplified in advance by high-fidelity PCR) were sequentially cloned in two different expression cassettes to generate a combinatorial library in which each clone could produce an rFab containing a certain VL-CL–VH-CH1 combination and all possible combinations occurring in individuals would be represented. Expression of the VL-CL and VH-CH1 cDNAs was under the control of identical promoters to obtain production of approximately equal levels of both rFab subunits. Both cDNAs were fused, at the amino-terminal end, with a bacterial sequence encoding a leader peptide which targets secretion of the HC and LC polypeptides to the periplasmic space, where they are assembled to generate a functional rFab. The VH-CH1 cDNA was also fused, at the carboxy-terminal end, with the cpIII-encoding gene of the filamen-
* Corresponding author. Mailing address for Paola Cattani: Istituto di Microbiologia, Universita` Cattolica del Sacro Cuore, Largo F. Vito 1, 00168 Rome, Italy. Phone: 39 (6) 3015.4336. Fax: 39 (6) 305.1152. Mailing address for: Gian Maria Rossolini: Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Universita` di Siena, Via Laterina 8, 53100 Siena, Italy. Phone: 39 (577) 26.3327. Fax: 39 (577) 26.3325. E-mail:
[email protected]. † This paper is dedicated to the memory of Giuseppe Satta, who passionately dedicated his life to investigation in the field of microbiological sciences. 1504
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FIG. 1. Schematic representation of the genetic vectors used for the construction of a combinatorial phage-display library of human rFabs (A) and for the production of soluble rFabs in E. coli cells (B). (A) The pCombIII phagemid vector was used for construction of the library as previously described (4, 35). For details, see Materials and Methods. LP, leader peptide; gIII, the cpIII-encoding gene of the filamentous phage. (B) Phagemids recovered after the last round of panning are modified to obtain the production of selected rFabs in a soluble form. For this purpose, the cpIII-encoding gene (gIII) is removed by digestion with SpeI and NheI and a genetic cassette encoding chloramphenicol resistance (cat) is inserted in its place. The antibiotic-resistance cassette has been engineered to include at each end a stop codon ( ) which goes in-frame with the VH-CH1 cDNA so that any insert orientation results in correct translation of the cDNA. The insertion of a selectable marker between the HC and LC expression cassettes helps to prevent the generation of deletion derivatives. In this way, E. coli clones which produce soluble rFabs reactive with the antigen used for panning are eventually obtained.
tous phage so that the HC polypeptide was actually produced as a fusion protein which could be incorporated at the tip of the filamentous phage by means of the cpIII moiety. The f1 origin of replication present in the vector is responsible for its ability to be packaged into filamentous phage particles. The combinatorial immunoglobulin fragment library was transformed into Escherichia coli XL-1 blue {recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 lac[F9 proAB lacIq lacZ M15 Tn10(Tetr)]; Stratagene} and converted into the phage-display format by infection of transformed bacteria with the interference-resistant helper phage VCSM13 (Stratagene) (Fig. 1A). When E. coli cells carrying the phagemid library are infected with an interference-resistant helper phage, each cell releases filamentous phage particles which contain as the genome the phagemid carried by that cell and which display at their tips the corresponding rFab. Immunoaffinity selection of clones producing HSV-specific rFabs from the library was performed by panning the library in its phage-display format for three rounds against an HSV antigen preparation bound to polystyrene wells (Behring
AG, Marburg, Germany) as previously described (7). After the third round of panning, eluted phage were converted to E. coli clones producing soluble rFabs by removal of the cpIII-encoding gene from the phagemid vector by digestion with SpeI and NheI. At the same time, a genetic cassette that has a stop codon at each end and encodes chloramphenicol resistance was inserted between the HC and LC expression cassettes to prevent the generation of deletion derivatives, which is sometimes observed with similar expression vectors due to recombination events between the two identical promoters (Fig. 1B). Crude preparations of soluble rFabs obtained from individual bacterial clones were then checked for HSV reactivity by enzyme-linked immunosorbent assays (ELISA). Crude preparations of soluble rFabs were obtained from E. coli clones grown aerobically at 37°C in SB medium (30 g of tryptone, 20 g of yeast extract, 10 g of MOPS [morpholinepropanesulfonic acid] per liter [pH 7]) containing chloramphenicol (170 mg/ml). Isopropyl-b-D-thiogalactopyranoside (1 mM) was added to cultures when they reached an A600 of 0.8 to 1, and growth was allowed
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TABLE 1. Sources of the 134 HSV clinical isolates analyzed in this study Site or type of specimen
Oral cavity Tongue Pharyngeal Eye Bronchoalveolar lavage Face (perioral)/neck Other cutaneous Genital Buttocks Perianal Urine Total
No. of isolates HSV-1
HSV-2
12 3 21 4 10 14 7
3 1
1 72
7 26 6 13 6 62
for a further 12 to 14 h. Cells were collected by centrifugation and resuspended in phosphate-buffered saline (PBS) (pH 7.2) (1/10 of the original culture volume). The cell suspension was subjected to three cycles of freezing and thawing and then centrifuged at 10,000 3 g for 10 min at 4°C. The cleared supernatant represented the crude preparation and was stored at 220°C in small aliquots until used. Enzyme immunoassays. The ELISA for rFab reactivity against HSV antigens was performed with commercial plates (Behring) according to the manufacturer’s instructions, with a peroxidase-conjugated anti-human IgG Fab-specific polyclonal antiserum (Sigma Chemical Co., St. Louis, Mo.) as the secondary antibody. Each rFab preparation, diluted 1:10 in PBS, was always assayed in duplicate in both HSV antigen-containing and control wells. Crude preparations of rFabs specific for human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) obtained from the same combinatorial antibody library (9) were also included as negative controls. Cells and viruses. Vero and HEp-2 cells (Eurobio, Les Ulis, France) were routinely used for culturing clinical specimens. Vero cells were also used for the propagation of HSV clinical isolates and reference strains. HEp-2 cells were also used for the propagation of adenovirus. MRC-5 cells (Eurobio) were used for the propagation of varicella-zoster virus (VZV) and HCMV. P3HR-1 cells (ATCC HTB 62) were used as a cell line to produce EBV antigens (37). Vero, HEp-2, and MRC-5 cells were grown in Eagle minimal essential medium (Gibco-Life Technologies Ltd., Paisley, Scotland) supplemented with 5% (vol/vol) heatinactivated fetal calf serum (FCS) and antibiotics (100 mg of streptomycin per ml and 100 U of penicillin G per ml). P3HR-1 cells were grown in RPMI 1640 medium (Gibco) supplemented with 10% (vol/vol) heat-inactivated FCS and antibiotics as described above. The production of EBV antigens by P3HR-1 cells was induced by treatment with O-tetradecanoylphorbol-13-acetate (TPA) as previously described (37). HSV-1 strain F (ATCC VR-733) and HSV-2 strain G (ATCC VR-734) were used as reference strains. One hundred thirty-four HSV clinical isolates (72 HSV-1 and 62 HSV-2 isolates [Table 1]) were analyzed in this study. They were from specimens received at the clinical virology laboratory of the Istituto di Microbiologia, UCSC, A. Gemelli Medical School, Rome, Italy, and were selected to represent a heterogeneous population of patients, including both inpatients from different clinical departments and outpatients from various geographical areas. Clinical specimens were routinely inoculated into Vero and HEp-2 cell monolayers, which were monitored every second day for the appearance of cytopathic effects. Identification and typing of viral isolates was performed with commercial type-specific monoclonal antibodies (Dako Diagnostics Ltd., Ely, United Kingdom) in direct IF assays. VZV strain Ellen (ATCC VR586), HCMV strain AD-169 (ATCC VR-538), and adenovirus strain PC-23 (a clinical isolate) were used as specificity controls. IF assays. IF assays were performed either with cell monolayers grown on glass slides or with scraped cells spotted onto glass slides. In the latter case, cell monolayers were washed once with PBS containing 2% (vol/vol) FCS, removed with a cell scraper, and transferred onto glass slides at a suitable cell density. Cells were fixed in cold acetone for 10 min. Direct IF assays with commercial monoclonal antibodies (Dako) were performed according to the manufacturer’s instructions. Indirect IF assays with rFabs were performed as follows. Fixed cells were incubated with crude rFab preparations (diluted 1:10 in PBS, unless otherwise specified) at 37°C for 45 min in a humid atmosphere, washed three times with PBS, incubated with a fluorescein isothiocyanate-conjugated anti-human IgG Fab-specific polyclonal antiserum (Sigma) at 37°C for 45 min in a humid atmosphere, washed again three times with PBS, and, after a final wash with distilled water, air dried. Slides were mounted in buffered glycerol and observed with a transmission fluorescence microscope (Zeiss, Jena, Germany). Slides prepared with MRC-5 cells infected with VZV or HCMV, TPA-stimulated
P3HR-1 cells, and HEp-2 cells infected with adenovirus were used as controls to check for rFab specificity. Nucleic acid sequencing. Nucleotide sequences were determined by the dideoxy chain termination method (25) with denatured double-stranded DNA templates, a Sequenase 2.0 DNA sequencing kit (Amersham International plc, Milan, Italy), and sequencing primers, seq-hG1 (59-AGAGGTGCTCTTGGAG GA) and seq-hk (59-ATAGAAGTTGTTCAGCAGGCA), designed on the constant HC and LC immunoglobulin regions, respectively, located downstream of the CDR3 variable domain. Plasmid DNA for sequencing was prepared by alkaline lysis, followed by acid-phenol extraction as previously described (30). Nucleotide sequence accession numbers. The nucleotide sequences determined here have been submitted to the EMBL/GenBank/DDBJ nucleotide sequence database and have been assigned accession no. X97767, X97481, X97482, and X97483.
RESULTS Crude soluble rFab preparations from 110 E. coli clones, selected from a combinatorial phage-display library of human rFabs after three rounds of panning against an HSV antigen preparation, were screened for ELISA reactivity toward the same HSV antigen preparation used for panning. Thirty-seven (33.6%) of the 110 rFabs showed specific reactivity and were further analyzed for the ability to recognize Vero cells infected by HSV-1 or HSV-2 reference strains in an indirect IF assay. Of the 37 ELISA-positive rFabs, 34 (91.9%) produced positive IF staining of HSV-infected cells. Of these 34 rFabs, 25 recognized cells infected by either HSV-1 or HSV-2, while the remaining 9 were apparently able to recognize only HSV-1infected cells (Fig. 2A and B and data not shown). No reactivity was observed when crude rFab preparations were tested, even undiluted, against uninfected cells (Fig. 2G and data not shown). Fourteen of the IF-positive rFabs (9 of the 25 showing typecommon reactivity and 5 of the 9 showing type 1-specific reactivity) apparently yielded brighter and more intense IF staining. Among them, two (rFab H37 [reactive with both types of HSV] and rFab H85 [reactive with HSV-1 only]) were randomly selected for further characterization. These two rFabs were tested in indirect IF assays at increasing dilutions (ranging from 1:10 to 1:2,560) against Vero cells infected by two reference HSV strains and against Vero and HEp-2 cells infected by 72 HSV-1 and 62 HSV-2 clinical isolates (Table 1). The crude preparation of rFab H37 gave a strong positive fluorescent signal with the F (HSV-1) and G (HSV-2) reference strains and with all the HSV-1 and HSV-2 clinical isolates tested, up to a dilution ranging from 1:320 to 1:1,280 (median, 1:640) (Fig. 2A and B and data not shown). The IF pattern observed with either viral type was mainly localized at the plasma membrane and at the more peripheral area of cells, sometimes appearing as diffused to the entire cell (Fig. 2A and B). The crude preparation of rFab H85 recognized the F (HSV-1) reference strain and all the HSV-1 isolates examined up to a dilution of 1:320 to 1:1,280 (median, 1:320), while even undiluted, it did not react with either the G (HSV-2) reference strain or any of the HSV-2 isolates tested (Fig. 2A and B and data not shown). Overall, the IF staining pattern was similar to that observed for rFab H37 (Fig. 2A). The IF staining pattern obtained with rFab H37 and rFab H85 was different from that observed with the type-specific commercial murine monoclonal antibodies used in this study (Fig. 2A and B), suggesting that the viral epitopes recognized by the former could be different from those recognized by the latter. The ability of rFab H37 to detect HSV-infected cells by indirect IF assay in Vero cell monolayers infected with either the F (HSV-1) or G (HSV-2) reference strain was investigated at different times after infection, and a comparison was also performed with commercial anti-HSV fluorescein-labeled murine monoclonal antibodies. With the crude rFab H37 prepa-
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FIG. 2. IF staining by crude rFab preparations and commercial type-specific anti-HSV monoclonal antibodies (Dako) of cells infected by HSV or other viruses or of uninfected cells. (A) HSV-1-infected Vero cells probed with rFab H37, rFab H85, and the HSV-1-specific murine monoclonal antibody (Dako) used for comparison (D1); (B) HSV-2-infected Vero cells probed with rFab H37, rFab H85, and the HSV-2-specific murine monoclonal antibody (Dako) used for comparison (D2); (C) VZV-infected MRC-5 cells probed with rFab H37 and rFab H85; (D) HCMV-infected MRC-5 cells probed with rFab H37 and rFab H85; (E) TPA-stimulated P3HR-1 cells probed with rFab H37 and rFab H85; (F) adenovirus-infected HEp-2 cells probed with rFab H37 and rFab H85; (G) Uninfected Vero cells probed with rFab H37 and rFab H85.
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TABLE 2. Detection of HSV-1 and HSV-2 infections by IF staining with either rFab H37 or commercial fluorescein-labeled HSV-specific monoclonal antibodies (Dako) at different times after infectiona No. of positive cultures/no. of cultures analyzed Time after infection (h)
6 12 18 24 30 48 a b
rFab H37
Commercial antibodies
HSV-1
HSV-2
HSV-1
HSV-2
0/4 3/4 3/4 4/4 4/4 4/4
0/4 2/4 3/4 3/4 4/4 4/4
0/4 0/4 2/4 3/4 3/4b 4/4
0/4 0/4 2/4 3/4 4/4 4/4
Typical cytopathic effects were evident at 24 to 30 h after infection. The negative culture did not show any evident cytopathic effects.
ration at a 1:10 dilution, a positive IF result was recorded with both viral types as early as 12 h after infection, when there was still no evidence of any cytopathic effect (Table 2). Overall, the detection times were shorter than those observed in the same experimental system with commercial anti-HSV monoclonal antibodies in direct IF assays (Table 2). No reactivity in IF assays was observed when crude preparations of rFab H37 and rFab H85, even undiluted, were tested against cells expressing antigens of VZV, HCMV, EBV, or adenovirus (Fig. 2C through F). To verify whether the cloned rFabs were different from those already described, the DNA sequences of the variable domains of rFab H37 and rFab H85 were determined. The deduced amino acid sequences of the HC variable domains of the two clones (Fig. 3) appear to be different from those previously reported for other human anti-HSV rFabs (6, 26). DISCUSSION Previous works have shown that it is possible to isolate from combinatorial phage-display antibody libraries E. coli clones producing human rFabs against a wide array of specificities, including epitopes of viral pathogens (4–8, 22, 35). While the greatest value of these reagents is obviously related to their potential use for in vivo immunotherapy or immunoprophylaxis (5, 7, 26, 27), they could also be useful for in vitro diagnostic purposes. In this work, starting from a combinatorial phage-display library of human IgG1(k) rFabs constructed from an individual who was seropositive for HSV-1, we selected several bacterial clones producing soluble rFabs reactive against either typecommon or type 1-specific HSV antigens. No clones producing HSV-2-specific rFabs could be selected from this library, as expected according to the serological profile of the donor. Characterization of two of the HSV-reactive rFabs, with one
showing a type-common reactivity and the other showing a type 1-specific reactivity, indicated that they could be reliably used for viral identification and typing in indirect IF assays. The specificity of these rFabs appeared to be the same as that of commercial murine monoclonal antibodies routinely used for HSV identification and typing, while the rFab showing type-common reactivity apparently allowed a higher-level detection sensitivity than that of commercial murine monoclonal antibodies used for comparison. This could be due to the use of an indirect IF assay for working with rFabs (21) but could also be related to a difference in the viral epitopes recognized by the two systems. Since indirect IF assays are more tedious and labor-intensive than are direct IF assays, it would also be of interest to produce fluorescein-labeled preparations of the rFabs mentioned above and evaluate their performances in direct IF assays. The major limit of a diagnostic system that consists of an HSV type-common reagent and an HSV-1-specific immunological reagent is the inability to discriminate between HSV-1 infections and mixed HSV-1–HSV-2 infections. However, dual HSV infections appear to be rare (13), as suggested by our experience. In the last 4 years, we have never found any dual infection in a total of 418 clinical samples positive for HSV and subject to viral typing. Since missing or modification of an epitope can occur with HSV isolates and cause false-negative results with monoclonal antibodies (14, 19, 20, 29), another limitation of our system could be its inability to correctly type HSV-1 isolates that lack the epitope recognized by rFab H85. However, the fact that this rFab was able to recognize all the HSV-1 strains analyzed in this study, including both a reference strain and 72 clinical isolates from heterogeneous specimens, suggests that the corresponding viral epitope is a rather conserved one and that false-negative typing results due to epitope variability should be rare in this case. A potential advantage of the diagnostic system based on the rFabs described above could be that crude preparations suitable for indirect IF assays are easily and inexpensively prepared from E. coli cultures in a clinical microbiology laboratory, with virtually no need for any additional equipment or competence other than those institutionally present. In this case, the cost of IF testing for HSV can be substantially reduced compared to that of commercial systems based on fluorescein-labeled murine monoclonal antibodies. It should be noted that the introduction of a selectable marker between the HC and LC expression cassettes in the vector for soluble rFab expression increased the stability of the expression system, resulting in a more reproducible rFab yield in crude preparations. Another potential advantage of similar rFabs produced in bacteria is that they can easily be engineered by recombinant DNA technology to obtain immunoconjugates which, carrying
FIG. 3. Amino acid sequences of the variable regions of rFab H37 and rFab H85 HC and LC. Framework (FR) and complementarity-determining (CDR) regions are indicated; the former are underlined.
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suitable reporter moieties, could be useful for diagnostic purposes in direct immunoassays. 17.
ACKNOWLEDGMENTS This work was supported in part by grant no. 94.01207.PF39 for Italian National Research Council (C.N.R.)-targeted project ACRO. Thanks are due to Teresa Mancini and Franca Pedone for excellent technical assistance.
18.
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