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II. Koper JW, Hagenaars AM. Notermans S. Prevention ofcross-reactions in the enzyme linked immunosorbent assay (ELISA) for the detection of Staphylococcus aureus enterotoxin type B in culture filtrates and foods. J Food Safety 1980;2:35-45. 12. Chang AH, Musser JM, Chow AW. A single clone which produces both TSST-I and SEA causes the majority of menstrual toxic shock syndrome. Clin Res 1991;39:36A. 13. Neill RJ, Fanning GR, Oelahoz F. WolffR, Gemski P. Oligonucleotide probes for detection and differentiation of Staphylococcus aureus
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strains containing genes for enterotoxins A, B, and C and toxic shock syndrome toxin 1. J Clin Microbiol 1990;28: 1514-8. 14. Johnson WM. Tyler SO, Ewan EP, Ashton FE, Pollard DR. Rozee KR. Detection of genes for enterotoxins, exfoliative toxins, and toxic shock syndrome toxin I in Staphylococcus aureus by the polymerase chain reaction. J Clin Microbiol 1991;29:426-30. IS. Recsei P, Kreiswirth B, O'Reilly M, Schlievert P, Gruss A, Novick R. Regulation of exoprotein gene expression by agr. Mol Gen Genet 1986;202: 58-6 I.
H. Martin Jones, Julius Schachter, and Richard S. Stephens
Departments ofPharmaceutical Chemistry and Laboratory Medicine and Francis I. Proctor Foundation. University ofCalifornia. San Francisco
The Chlamydia trachomatis immunodominant major outer membrane protein (MOMP) is both a target of neutralizing antibodies and the serotyping antigen and thus has been a focus of diagnostic, seroepidemiologic, and experimental investigations. The microimmunofluorescence (MIF) test has been the principal tool in serologic investigations of chlamydial infections but is difficult and expensive for routine use; moreover, since it uses whole organisms as antigen, it is incapable of revealing the molecular specificity of the humoral response to infection. These limitations were resolved by using synthetic peptides corresponding to serovar-specific antigenic regions of MOMP in an ELISA-based format to analyze the serospecificity of sera from trachoma cases. The ELISA reaction to the surface-exposed MOMP sequence variable segment 1 was immunodominant and serovar-specific and was in concordance with serovar specificity according to paired MIF test determinations. Understanding the patterns of humoral responses to MOMP determinants in patient populations will advance our knowledge of their role in the immunobiology of naturally acquired infection. Chlamydia trachomatis is an intracellular bacterial parasite causing a wide spectrum of oculogenital diseases worldwide. It remains the leading cause of preventable blindness in the world and of sexually transmitted bacterial disease (STD) in the United States. Isolation or direct detection methods often fail to identify the presence of C trachomatis organisms at chronic disease stages; thus, the serologic response may be the only demonstration of a disease's chlamydial origin. Serologic evaluations have been a key factor in demonstrating the wide spectrum of diseases of chlamydial etiology as well as shared pathogenic mechanisms ofchlamydial infections.
Received 20 February 1992: revised 19 May 1992. Grant support: National Institutes of Health (EY-07757, AI-21912, and AI-31499). Reprints or correspondence: Dr. Richard S. Stephens, Francis I. Proctor Foundation, University of California, San Francisco, CA 94143-0412. The Journal of Infectious Diseases 1992;166:915-9 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6604-0036$01.00
The serologic response to natural and experimental C trachomatis infection has been monitored by the microirnmunofluorescence (MIF) test, which uses fixed whole organisms as antigen and is capable of serotype resolution only at endpoint dilutions of antisera [I]. However, this assay is technically difficult, subjective, and labor intensive and requires extensive facilities and skilled personnel, making it impractical for field use or for large-scale, rapid screening of clinical or experimental samples. Moreover, only a few laboratories worldwide are equipped to do the assay routinely, so all field and most clinical samples must await transport to a central facility for analysis. We report the successful application to clinical samples of an ELISA-based serologic immunoassay incorporating, as antigens, synthetic peptides duplicating the serovar-specific regions of the major outer membrane protein (MOMP) ofC trachomatis. The MOMP is an antigenically complex protein with surface regions having serovar specificity for each of the distinct serovars. It also has cross-reactive regions with subspecies- and species-specificdeterminants [2]. On the basis of
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Evaluation of the Humoral Immune Response in Trachoma to Chlamydia trachomatis Major Outer Membrane Proteins by Sequence-Defined Immunoassay
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Concise Communications
Methods SDI parameters. Peptides corresponding to the inferred amino acid sequence of the MOMP VS1 (amino acids 64-80) and VS2 (amino acids 139-152; figure 1) for serovars A, B, Ba, C, D, and E were synthesized using published sequences [5] and were used without further purification. The peptides were dissolved to 3 mg/mL in distilled water containing 0.025% sodium azide and stored in silicone-coated glass vials at 4°C. Polystyrene microtiter plates (Immulon 2; Dynatech, Chantilly, VA) were coated with 50 JLL of 100 pmol of peptide/well [4] in 50 mM bicarbonate buffer, pH 9.6, overnight at 37°C. This peptide concentration was found to be optimal by block titration with control antisera from rabbits. After adsorption, the wells were blocked with freshly prepared 5% bovine serum albumin in PBS
for 1 h at 37°C. Serial dilution of antisera does not change the relative serologic specificities to the peptides (unpublished data); thus, the standard SDI used human sera at a dilution of 1/128 in PBScontaining 0.05%Tween 20 (PBS-T). Sera diluted 1/128 that had initially nonreactive or equivocal SDI responses were reevaluated at a dilution of 1/32. Antigenicity of each peptide was confirmed immunologically using polyclonal anti-elementary body and peptide-specificantisera or monoclonal antibodies. Antisera were diluted in PBS-T and incubations were for 1 h each at 37°C. The dilution of the anti-human IgG-horseradish peroxidase conjugate was determined by block titration using human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) as antigen. Before and after addition of horseradish peroxidase-conjugated antihuman IgG (Zymed Laboratories, South San Francisco) or the appropriate animal sera at an optimal dilution (usually 1/1000), the wells were washed three times with PBS-T. Before addition of substrate (0.1 % hydrogen peroxide) and chromagen (1 mg/ mL o-phenylenediamine; Sigma, St. Louis) in PBS, the wells were rinsed twice with PBS. The color was allowed to develop for 15 min in the dark at room temperature and the reaction terminated by the addition of 25 JLL of 8 N H2S04 , The optical density at 492 nm was measured on a Titertek Multiscan (Flow Laboratories, McLean, VA) ELISA plate reader with commercially available software. Reactivity of sera from trachoma patients by SDI and MIF. Human sera were obtained from a collection of samples from a trachoma-endemic region of Africa [6] and were stored frozen until used. These were serotyped in a masked evaluation using the MIF test. Control sera were obtained locally and were negative by MIF. SDI seroreactivity criteria. Reactivities of a given serum to each peptide were compared with the averaged optical density values of each peptide for four MIF-negative control sera. A positive response to any peptide had to exceed twice that of the control value. To be considered specific to a particular peptide, the response had to exceed twice the value of the positive responses to the other peptides. lethe positive responsesofa particular sample did not differ by the required amount, the serum was judged to be bi- or multispecific, depending on the number of positive anti-peptide responses.
Results Serovar
VSl
VS2
A
APTTSDVAGLEKDPVAN
TKTQSSGFDTANIV
B
AKPTTTTGNAVAPSTLT
NNENQTKVSNGAFV
Ba
AKPTATTGNATAPSTLT
NNENQTKVSNSTFV
C
APTTSDVAGLQNDPTTN
TKTQSSSFNTAKLI
D
AKPTTDTGNSAAPSTLT
DNENQKTVKAESV
E
DKPTSTTGNATAPTTLT
DNENQSTVKTNSV
Figure 1. Amino acid sequences of MOMP variable segment (VS) synthetic peptides. All peptides include an amino-terminal cysteine and are numbered 64-80 for VS1 and 139-152 for VS2 according to Yuan et at. [5].
We synthesized VS I and VS2 for each of the trachoma serovars (figure I) on the basis of the following data and rationale. When fusion proteins incorporating Band C VS I and VS2 regions of MOMP were used as antigens, VSI was shown to be immunodominant in rabbits immunized with B or C elementary bodies [7]. Synthetic VS 1 peptides probed with antisera from experimentally immunized animals exhibited similar reactivity (data not shown), and more important, several samples of human sera with known MIF titers and specificities reacted specifically with the homologous VS I synthetic peptide, resulting in serovar specificity identical to the MIF determination. However, since serovar-specific monoclonal antibodies may bind either VSI or VS2 regions
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MOMP gene sequencing of different serovars, four polymorphic sequence regions of the MOMP called variable segments (VS) 1-4 have been identified [3]. Mapping of monoclonal antibody reactivities has shown that VS I and VS2 are capable of binding serovar-specific monoclonal antibodies. The MOMP is also the target of neutralizing antibodies and therefore, in addition to being the major serotyping antigen, has potential vaccine applications. Thus, serologic assessments may be useful in the design and development of an effective vaccine in that specific anti-MOMP responses in natural infections can be determined and, after immunity develops, correlated with resistance. Since our ELISA-based approach can exclude any or all cross-reactive epitopes, is economical, and uses readily standardized compounds of known composition and purity, it has both a considerable practical advantage over the MIF test and the requisite resolution on a molecular scale. This method, the sequence-defined immunoassay (SDI), also known as "site-directed" serology [4], has not been applied to the human humoral response to pathogens other than viruses. This may in part be because of logistic complications arising from the large number of recognized serovariants, complexity of antigens, or the lack of a readily identifiable, uniformly expressed, stable serotyping antigen.
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110 1992;166 (October)
ologous VS2 peptide, resulting in A and C bispecificity by our definition in both. Most of the SDI and MIF results were in concordance (figure 2). The only discrepancy was that 5 of the serotype A samples by SDI were judged to be A and C bispecific by MIF and 2 samples A and C bispecific by SDI were either A or C by MIF. As a negative control, reactivity of the trachoma samples to the STD serovar peptides specific for serovars D and E were low, although 1 serovar B sample was not tested to D and E peptides because not enough serum was available. Likewise, sera from STD patients that were positive by MIF did not react to the trachoma-specific peptides (data not shown).
Discussion Seroepidemiologic studies have contributed significantly to our understanding of the spectrum of symptomatic and asymptomatic diseases associated with C. trachomatis. Serology has played an important role in the diagnosis of infant pneumonia ofchlamydial origin and lymphogranuloma venereum [10]. The use ofSDI would greatly benefit these investigations since it is an important practical advance over current methods. We have shown by SDI that the VSl region of MOMP is immunodominant and results in type-specific determina-
16 -r----------------------------......, 14 12 10
Figure 2. Reactivities of sera from trachoma patients to variable segment 1 (A) and 2 (B) synthetic peptides from serovars A, B, C, Ba, D, and E (bars for each sample, left to right). Serovar B sample assayed only with A, B, and C peptides and 7 nonreactive samples were omitted. Microimmunofluorescence (MIF) determinations are at bottom. Reactivities are signal-to-noise ratios (SjN) calculated for each serum by dividing optical density response to given peptide by averaged optical density values of 4 MIF-negative control sera. *, sample diluted 1/32; all others were assayed at 1/128. t, MIFwasnonspecific.
SIN
8 6
4
1
2
3
4
5
6
7
8
9 10 11 12 13* 14 15 16 17 18* 19 20 21 22 23 24* 25
16-r----------------------------......, SIN
4 2 0
MW{
2
3
4
6
7
8
9 10 11 12 13* 14 15 16 17 18* 19 20 21 22 23 24* 25
A C
A
A A A C C C
A
A
A
1
5
A
A
A B A A C Ba
A C C B D E
A C
t
C A A C
C
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of MOMP [8, 9], then either of these VS regions was potentially serologically significant. Most samples of human sera from inhabitants of a trachoma-endemic region of Tunisia exhibited such a strikingly specific and sensitive anti-peptide activity that it was possible to categorize them as to serotype by visual inspection alone at a serum dilution of 1/128. Figure 2 shows that regardless of serogroup, virtually all reactivity was confined to the VS1 region; this reactivity allowed us to assign serovar specificity to the serologic response of most of the patients. Note that not all samples from a population will show a specific response regardless of which assay methods or parameters are chosen. Of the 33 samples (including those assayed at 1/32 dilution), 17 were serotype A, 2 were serotype B, 4 were serotype C, 2 were A and C bispecific, 1 was multispecific, and 7 had no values greater than twice that of control (i.e., nonreactive). The group nonreactive by SDI were either not reactive by MIF or were broadly cross-reactive, which implies responses to a different antigen (such as lipopolysaccharide) or a subspecies- or species-specific VS4 determinant and/or a VS1 specificity not included in the current SDI format. A positive VS2 reactivity was present in roughly onehalf of the samples and was typically only just positive and also specific for the homologous VSl reactivity. In only 2 samples (numbers 20 and 25) the reactivities were to a heter-
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dilution determinations). This assay format is a proven serologic tool and will be of great value in future clinical screening applications and field trials. Possibly with further development (epitope selection and combinations, isotyping the response), this approach may have diagnostic value for chlamydia1infections and those due to other pathogenic microorganisms that have sequence-variant surface antigens. Diagnostic applications must first be established in situations in which serologic analysis by MIF has been similarly applied. These include lymphogranuloma venereum and infant pneumonia [10] and the demonstration of antibody in tears as specific for trachoma [14, 15]. The main limitation to chlamydial serology has been the cumbersome nature of the MIF assay; this objection is mitigated by SDI. Additionally, and perhaps most important, the identification of the chlamydial MOMP immunoreactive and immunoprotective determinants in individuals during naturally acquired infection will broaden our understanding of the factors involved in transmission ofand resistance to this organism and may facilitate decisions regarding public health policy and vaccine development. Acknowledgments
We gratefully appreciate the technical assistance of Jeanne Moncada. References I. Wang SP. Grayston JT. Immunologic relationship between genital TRIC, lymphogranuloma venereum, and related organisms in a new microtiter indirect immunofluorescence test. Am J Ophthalmol
1970;70:367-74. 2. Stephens RS, Tam MR, Kuo CC, Nowinski RC. Monoclonal antibodies to Chlamydia trachotnatis:antibody specificities and antigen characterization. J Immunol 1982;128: 1083-9. 3. Stephens RS. Sanchez-Pescador R. Wagar EA. Inouye C, Urdea M. Diversity of Chlamydia trachontatis major outer membrane protein genes. J Bacteriol 1987; 169:3879-85. 4. Norrby E. Biberfield G, Chiodi F, et al. Discrimination between antibodies to HIV and to related retroviruses using site-directed serology. Nature 1987;329:248-50. 5. Yuan Y. Zhang YX. Watkins N. Caldwell HD. Nucleotide and deduced amino acid sequences for the four variable domains of the major outer membrane proteins of the 15 Chlamydia trachomatis serovars. Infect Immun 1989;57: 1040-9. 6. Hanna L. Jawetz E. Briones O. et al. Antibodies to TRIC agents in tears and serum of naturally infected humans. J Infect Dis 1973; 127:
95-8. 7. Jones HM. Volpicelli M. Stephens RS. Immunoassay of polyclonal antibody responses to Chlamydia trachomatis variable segment one and variable segment two major outer membrane protein peptides. In: Bowie WR. Caldwell HD. Jones RP, et al., eds. Chlamydial infections: proceedings of the seventh international symposium on human chlamydial infections. New York: Cambridge University Press.
1990: 109-12. 8. Baehr W. Zhang YX. Joseph T, et al. Mapping antigenic domains expressed by Chlamydia trachomatis major outer membrane protein genes. Proc Nat! Acad Sci USA 1988;85:4000-4. 9. Stephens RS. Wagar EA. Schoolnik GK. High-resolution mapping of
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tions for sera obtained from a trachoma-endemic region. The preponderance of serotypes A and C in this population has been noted previously using MIF [6]. Even though our assay results and previous MIF data on the same population showed this strong C-complex representation (serovars A and C) in the trachoma patient samples, several B-complex sera among these were detected. Moreover, in preliminary evaluations with an identical experimental format of sera from patients with STDs, we could easily detect B-complex representatives (B- and E-type-specific responses; unpublished data). Thus, we believe that the SDI can be applied to both B- and C-complex serogroups. Some of the samples were not typeable by either MIF or SDI, perhaps because of sequence variability within given serovars, the existence of new, uncharacterized serovars [II ], or a low-titered response to multiple serovars. Closely related serovars, such as serovars C and J, Band Ba, G and F, or D and E, cannot be distinguished by one-way MIF assay. For example, the only difference between Band Ba is that antisera to Ba shows greater cross-reactivity with other serovars than does antisera to B. From a practical point of view, these are typically not discriminated in MIF evaluations, and this assay is often simplified by the pooling ofantigens in a target population-dependent manner. Our finding of dominant serologic responses to VS 1 may explain in part the difficulty in discriminating between closely related serovars. The VS I sequences for C and J and for G and F are identical for each pair, and Band Ba have two relatively conserved amino acid differences. Complications may also arise from humoral responses to infection simultaneously or sequentially by multiple serovars; the latter may result in an "original antigenic sin" phenomenon in which a boost with an antigen closely related to the priming antigen can elicit a strong B cell memory response to the priming antigen [12]. It was surprising that the VS2 region was not very reactive with these sera, especially since VS2 is the most sequencevariable segment. This may reflect the C-complex bias of the sample population, or this region may be involved in a more conformational epitope that would not necessarily be detected with these short linear peptides. Evidence does exist for its surface exposure in both Band C subgroups [13] (unpublished data), and serovar-specific monoclonal antibodies have been mapped to this region [8, 9]. The precise role of the anti-VSI response in chlamydial immunobiology is not yet known, and its relationship with more complicated, conformational determinants remains to be established. The SDI results in this trachoma-endemic population mimicked the results of MIF. If this concordance holds true for the serovariants that cause STDs, then the utility ofthe assay will be universal for serologic investigations of diseases of chlamydial origin. We propose that SDI is an effective alternative to MIF, and it has important practical advantages over MIF (flexibility, simplicity, low cost, objectivity, portability, and single-
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110 1992; 166 (October)
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serovar-specific and common antigenic determinants of the major outer membrane protein of Chlamydia trachomatis. J Exp Med 1988; 167:817-31. 10. Schachter J, Dawson CR. Human chlamydial infections. Littleton, MA: PSG Publishing. 1978. II. Dean D. Patton M, Stephens RS. Direct sequence evaluation of the major outer membrane protein gene variant regions of Chlamydia trachomatis subtypes D'. 1', and L2'. Infect Immun 1991;59: 157982. 12. Fazekas de St Groth S. Webster RG. Disquisitions on original antigenic sin. II. Proof in lower creatures. J Exp Med 1966; 124:347-6 I.
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13. Su H. Zhang YX. Barrera O. Watkins NO. Caldwell HD. Differential effect of trypsin on infectivity of Chlamydia trachomatis:loss of'infectivity requires cleavage of major outer membrane protein variable domains II and IV. Infect Immun 1988;56:2094-100. 14. Treharne JD. Dwyer RSC, Darougar S. Jones BR. Daghfous T. Antichlamydial antibody in tears and sera and serotypes of Chlamydia trachomatis isolated from school children in southern Tunisia. Br J Ophthalmol 1978;62:509-15. 15. Taylor HR, Siler JA, Mkocha HA, et at. Longitudinal study of the microbiology of endemic trachoma. J Clin Microbiol 1991;29: 1593-5.
Pierre J. Plourde, Mark Tyndall, Elizabeth Agoki, John Ombette, Leslie A. Slaney, Lourdes J. D'Costa, Jackoniah O. Ndinya-Achola, and Francis A. Plummer
World Health Organization Centre for Research and Training on Sexually Transmitted Diseases, Department of Medical Microbiology. University of Nairobi, and Special Treatment Clinic, Nairobi City Commission, Kenya; Departments ofInternal Medicine and Medical Microbiology, University ofManitoba, Canada
Sexually transmitted diseases (STDs) have had a significant adverse effect on reproductive and child health worldwide. The control of STDs such as gonorrhea is therefore an absolute priority. Cefixime, an oral third-generation cephalosporin with in vitro activity similar to that of ceftriaxone, may be an effective candidate for the treatment of gonorrhea. The efficacy of a single oral 400-mg dose of cefixime was compared with that of a single intramuscular 250-mg dose of ceftriaxone for the treatment of Neisseria gonorrhoeae urethritis in 190 men and cervicitis in 46 women in Nairobi, Kenya. A bacteriologic cure was recorded in 100% of63 evaluatable patients treated with ceftriaxone and 118 (98%) of 121 evaluatable patients treated with cefixime. Cefixime, as a single oral dose, is an effective alternative for the treatment of uncomplicated gonococcal urethritis in men and cervicitis in women.
Sexually transmitted diseases (STDs), including gonorrhea, have had a significant adverse effect on reproductive and child health worldwide. Slowing the spread ofgonorrhea is a major public health priority at this time. Treatment of Neisseria gonorrhoeae infection has been complicated by the
Received 3 February 1992; revised 4 May 1992. Presented in part: Ninth meeting of the International Society for Sexually Transmitted Diseases Research. Banff, Canada, October 1991. This study received institutional review board approval from the University of Manitoba and the University of Nairobi. Verbal informed consent was obtained prior to enrollment into the study. Financial support: American Cyanamid Company, Lederle Laboratories; Medical Research Council ofCanada (fellowships to PJ.P. and M.T., Scientist award to EA.P.). Reprints or correspondence: Dr. Pierre J. Plourde, Department of Medical Microbiology. Basic Sciences Building. Room 544. 730 William Ave., Winnipeg. Manitoba. Canada. R3E OW3. The Journal of Infectious Diseases 1992;166:919-22 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6604-0037$01.00
emergence of chromosomally and plasmid-mediated antimicrobial-resistant strains [1]. Plasmid-mediated resistance, first reported in 1976. resulted in the emergence of penicillinase-producing N. gonorrhoeae [2]. Third-generation cephalosporins offer an excellent therapeutic choice because of their intrinsic .B-lactamase resistance. Intramuscular ceftriaxone has been established as an ideal treatment for N. gonorrhoeae [3] and is currently the recommended first-line therapy of choice [4]. However, wherever possible, it would be preferable to avoid parenteral therapy when an efficacious oral antibiotic exists. Cefixime, an orally administered third-generation cephalosporin, has favorable phamacokinetics for single-dose treatment of uncomplicated gonorrhea [5] and has high in vitro activity against antimicrobial-resistant isolates of N. gonorrhoeae [6,7]. This study was designed to determine the safety and efficacy of a single 400-mg oral dose of cefixime compared with a single 250-mg intramuscular (im) dose of ceftriaxone for
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Single-Dose Cefixime versus Single-Dose Ceftriaxone in the Treatment of Antimicrobial-Resistant Neisseria gonorrhoeae Infection
Concise Communications
II. Koper JW, Hagenaars AM. Notermans S. Prevention ofcross-reactions in the enzyme linked immunosorbent assay (ELISA) for the detection of Staphylococcus aureus enterotoxin type B in culture filtrates and foods. J Food Safety 1980;2:35-45. 12. Chang AH, Musser JM, Chow AW. A single clone which produces both TSST-I and SEA causes the majority of menstrual toxic shock syndrome. Clin Res 1991;39:36A. 13. Neill RJ, Fanning GR, Oelahoz F. WolffR, Gemski P. Oligonucleotide probes for detection and differentiation of Staphylococcus aureus
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strains containing genes for enterotoxins A, B, and C and toxic shock syndrome toxin 1. J Clin Microbiol 1990;28: 1514-8. 14. Johnson WM. Tyler SO, Ewan EP, Ashton FE, Pollard DR. Rozee KR. Detection of genes for enterotoxins, exfoliative toxins, and toxic shock syndrome toxin I in Staphylococcus aureus by the polymerase chain reaction. J Clin Microbiol 1991;29:426-30. IS. Recsei P, Kreiswirth B, O'Reilly M, Schlievert P, Gruss A, Novick R. Regulation of exoprotein gene expression by agr. Mol Gen Genet 1986;202: 58-6 I.
H. Martin Jones, Julius Schachter, and Richard S. Stephens
Departments ofPharmaceutical Chemistry and Laboratory Medicine and Francis I. Proctor Foundation. University ofCalifornia. San Francisco
The Chlamydia trachomatis immunodominant major outer membrane protein (MOMP) is both a target of neutralizing antibodies and the serotyping antigen and thus has been a focus of diagnostic, seroepidemiologic, and experimental investigations. The microimmunofluorescence (MIF) test has been the principal tool in serologic investigations of chlamydial infections but is difficult and expensive for routine use; moreover, since it uses whole organisms as antigen, it is incapable of revealing the molecular specificity of the humoral response to infection. These limitations were resolved by using synthetic peptides corresponding to serovar-specific antigenic regions of MOMP in an ELISA-based format to analyze the serospecificity of sera from trachoma cases. The ELISA reaction to the surface-exposed MOMP sequence variable segment 1 was immunodominant and serovar-specific and was in concordance with serovar specificity according to paired MIF test determinations. Understanding the patterns of humoral responses to MOMP determinants in patient populations will advance our knowledge of their role in the immunobiology of naturally acquired infection. Chlamydia trachomatis is an intracellular bacterial parasite causing a wide spectrum of oculogenital diseases worldwide. It remains the leading cause of preventable blindness in the world and of sexually transmitted bacterial disease (STD) in the United States. Isolation or direct detection methods often fail to identify the presence of C trachomatis organisms at chronic disease stages; thus, the serologic response may be the only demonstration of a disease's chlamydial origin. Serologic evaluations have been a key factor in demonstrating the wide spectrum of diseases of chlamydial etiology as well as shared pathogenic mechanisms ofchlamydial infections.
Received 20 February 1992: revised 19 May 1992. Grant support: National Institutes of Health (EY-07757, AI-21912, and AI-31499). Reprints or correspondence: Dr. Richard S. Stephens, Francis I. Proctor Foundation, University of California, San Francisco, CA 94143-0412. The Journal of Infectious Diseases 1992;166:915-9 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6604-0036$01.00
The serologic response to natural and experimental C trachomatis infection has been monitored by the microirnmunofluorescence (MIF) test, which uses fixed whole organisms as antigen and is capable of serotype resolution only at endpoint dilutions of antisera [I]. However, this assay is technically difficult, subjective, and labor intensive and requires extensive facilities and skilled personnel, making it impractical for field use or for large-scale, rapid screening of clinical or experimental samples. Moreover, only a few laboratories worldwide are equipped to do the assay routinely, so all field and most clinical samples must await transport to a central facility for analysis. We report the successful application to clinical samples of an ELISA-based serologic immunoassay incorporating, as antigens, synthetic peptides duplicating the serovar-specific regions of the major outer membrane protein (MOMP) ofC trachomatis. The MOMP is an antigenically complex protein with surface regions having serovar specificity for each of the distinct serovars. It also has cross-reactive regions with subspecies- and species-specificdeterminants [2]. On the basis of
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
Evaluation of the Humoral Immune Response in Trachoma to Chlamydia trachomatis Major Outer Membrane Proteins by Sequence-Defined Immunoassay
916
Concise Communications
Methods SDI parameters. Peptides corresponding to the inferred amino acid sequence of the MOMP VS1 (amino acids 64-80) and VS2 (amino acids 139-152; figure 1) for serovars A, B, Ba, C, D, and E were synthesized using published sequences [5] and were used without further purification. The peptides were dissolved to 3 mg/mL in distilled water containing 0.025% sodium azide and stored in silicone-coated glass vials at 4°C. Polystyrene microtiter plates (Immulon 2; Dynatech, Chantilly, VA) were coated with 50 JLL of 100 pmol of peptide/well [4] in 50 mM bicarbonate buffer, pH 9.6, overnight at 37°C. This peptide concentration was found to be optimal by block titration with control antisera from rabbits. After adsorption, the wells were blocked with freshly prepared 5% bovine serum albumin in PBS
for 1 h at 37°C. Serial dilution of antisera does not change the relative serologic specificities to the peptides (unpublished data); thus, the standard SDI used human sera at a dilution of 1/128 in PBScontaining 0.05%Tween 20 (PBS-T). Sera diluted 1/128 that had initially nonreactive or equivocal SDI responses were reevaluated at a dilution of 1/32. Antigenicity of each peptide was confirmed immunologically using polyclonal anti-elementary body and peptide-specificantisera or monoclonal antibodies. Antisera were diluted in PBS-T and incubations were for 1 h each at 37°C. The dilution of the anti-human IgG-horseradish peroxidase conjugate was determined by block titration using human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) as antigen. Before and after addition of horseradish peroxidase-conjugated antihuman IgG (Zymed Laboratories, South San Francisco) or the appropriate animal sera at an optimal dilution (usually 1/1000), the wells were washed three times with PBS-T. Before addition of substrate (0.1 % hydrogen peroxide) and chromagen (1 mg/ mL o-phenylenediamine; Sigma, St. Louis) in PBS, the wells were rinsed twice with PBS. The color was allowed to develop for 15 min in the dark at room temperature and the reaction terminated by the addition of 25 JLL of 8 N H2S04 , The optical density at 492 nm was measured on a Titertek Multiscan (Flow Laboratories, McLean, VA) ELISA plate reader with commercially available software. Reactivity of sera from trachoma patients by SDI and MIF. Human sera were obtained from a collection of samples from a trachoma-endemic region of Africa [6] and were stored frozen until used. These were serotyped in a masked evaluation using the MIF test. Control sera were obtained locally and were negative by MIF. SDI seroreactivity criteria. Reactivities of a given serum to each peptide were compared with the averaged optical density values of each peptide for four MIF-negative control sera. A positive response to any peptide had to exceed twice that of the control value. To be considered specific to a particular peptide, the response had to exceed twice the value of the positive responses to the other peptides. lethe positive responsesofa particular sample did not differ by the required amount, the serum was judged to be bi- or multispecific, depending on the number of positive anti-peptide responses.
Results Serovar
VSl
VS2
A
APTTSDVAGLEKDPVAN
TKTQSSGFDTANIV
B
AKPTTTTGNAVAPSTLT
NNENQTKVSNGAFV
Ba
AKPTATTGNATAPSTLT
NNENQTKVSNSTFV
C
APTTSDVAGLQNDPTTN
TKTQSSSFNTAKLI
D
AKPTTDTGNSAAPSTLT
DNENQKTVKAESV
E
DKPTSTTGNATAPTTLT
DNENQSTVKTNSV
Figure 1. Amino acid sequences of MOMP variable segment (VS) synthetic peptides. All peptides include an amino-terminal cysteine and are numbered 64-80 for VS1 and 139-152 for VS2 according to Yuan et at. [5].
We synthesized VS I and VS2 for each of the trachoma serovars (figure I) on the basis of the following data and rationale. When fusion proteins incorporating Band C VS I and VS2 regions of MOMP were used as antigens, VSI was shown to be immunodominant in rabbits immunized with B or C elementary bodies [7]. Synthetic VS 1 peptides probed with antisera from experimentally immunized animals exhibited similar reactivity (data not shown), and more important, several samples of human sera with known MIF titers and specificities reacted specifically with the homologous VS I synthetic peptide, resulting in serovar specificity identical to the MIF determination. However, since serovar-specific monoclonal antibodies may bind either VSI or VS2 regions
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MOMP gene sequencing of different serovars, four polymorphic sequence regions of the MOMP called variable segments (VS) 1-4 have been identified [3]. Mapping of monoclonal antibody reactivities has shown that VS I and VS2 are capable of binding serovar-specific monoclonal antibodies. The MOMP is also the target of neutralizing antibodies and therefore, in addition to being the major serotyping antigen, has potential vaccine applications. Thus, serologic assessments may be useful in the design and development of an effective vaccine in that specific anti-MOMP responses in natural infections can be determined and, after immunity develops, correlated with resistance. Since our ELISA-based approach can exclude any or all cross-reactive epitopes, is economical, and uses readily standardized compounds of known composition and purity, it has both a considerable practical advantage over the MIF test and the requisite resolution on a molecular scale. This method, the sequence-defined immunoassay (SDI), also known as "site-directed" serology [4], has not been applied to the human humoral response to pathogens other than viruses. This may in part be because of logistic complications arising from the large number of recognized serovariants, complexity of antigens, or the lack of a readily identifiable, uniformly expressed, stable serotyping antigen.
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ologous VS2 peptide, resulting in A and C bispecificity by our definition in both. Most of the SDI and MIF results were in concordance (figure 2). The only discrepancy was that 5 of the serotype A samples by SDI were judged to be A and C bispecific by MIF and 2 samples A and C bispecific by SDI were either A or C by MIF. As a negative control, reactivity of the trachoma samples to the STD serovar peptides specific for serovars D and E were low, although 1 serovar B sample was not tested to D and E peptides because not enough serum was available. Likewise, sera from STD patients that were positive by MIF did not react to the trachoma-specific peptides (data not shown).
Discussion Seroepidemiologic studies have contributed significantly to our understanding of the spectrum of symptomatic and asymptomatic diseases associated with C. trachomatis. Serology has played an important role in the diagnosis of infant pneumonia ofchlamydial origin and lymphogranuloma venereum [10]. The use ofSDI would greatly benefit these investigations since it is an important practical advance over current methods. We have shown by SDI that the VSl region of MOMP is immunodominant and results in type-specific determina-
16 -r----------------------------......, 14 12 10
Figure 2. Reactivities of sera from trachoma patients to variable segment 1 (A) and 2 (B) synthetic peptides from serovars A, B, C, Ba, D, and E (bars for each sample, left to right). Serovar B sample assayed only with A, B, and C peptides and 7 nonreactive samples were omitted. Microimmunofluorescence (MIF) determinations are at bottom. Reactivities are signal-to-noise ratios (SjN) calculated for each serum by dividing optical density response to given peptide by averaged optical density values of 4 MIF-negative control sera. *, sample diluted 1/32; all others were assayed at 1/128. t, MIFwasnonspecific.
SIN
8 6
4
1
2
3
4
5
6
7
8
9 10 11 12 13* 14 15 16 17 18* 19 20 21 22 23 24* 25
16-r----------------------------......, SIN
4 2 0
MW{
2
3
4
6
7
8
9 10 11 12 13* 14 15 16 17 18* 19 20 21 22 23 24* 25
A C
A
A A A C C C
A
A
A
1
5
A
A
A B A A C Ba
A C C B D E
A C
t
C A A C
C
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of MOMP [8, 9], then either of these VS regions was potentially serologically significant. Most samples of human sera from inhabitants of a trachoma-endemic region of Tunisia exhibited such a strikingly specific and sensitive anti-peptide activity that it was possible to categorize them as to serotype by visual inspection alone at a serum dilution of 1/128. Figure 2 shows that regardless of serogroup, virtually all reactivity was confined to the VS1 region; this reactivity allowed us to assign serovar specificity to the serologic response of most of the patients. Note that not all samples from a population will show a specific response regardless of which assay methods or parameters are chosen. Of the 33 samples (including those assayed at 1/32 dilution), 17 were serotype A, 2 were serotype B, 4 were serotype C, 2 were A and C bispecific, 1 was multispecific, and 7 had no values greater than twice that of control (i.e., nonreactive). The group nonreactive by SDI were either not reactive by MIF or were broadly cross-reactive, which implies responses to a different antigen (such as lipopolysaccharide) or a subspecies- or species-specific VS4 determinant and/or a VS1 specificity not included in the current SDI format. A positive VS2 reactivity was present in roughly onehalf of the samples and was typically only just positive and also specific for the homologous VSl reactivity. In only 2 samples (numbers 20 and 25) the reactivities were to a heter-
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dilution determinations). This assay format is a proven serologic tool and will be of great value in future clinical screening applications and field trials. Possibly with further development (epitope selection and combinations, isotyping the response), this approach may have diagnostic value for chlamydia1infections and those due to other pathogenic microorganisms that have sequence-variant surface antigens. Diagnostic applications must first be established in situations in which serologic analysis by MIF has been similarly applied. These include lymphogranuloma venereum and infant pneumonia [10] and the demonstration of antibody in tears as specific for trachoma [14, 15]. The main limitation to chlamydial serology has been the cumbersome nature of the MIF assay; this objection is mitigated by SDI. Additionally, and perhaps most important, the identification of the chlamydial MOMP immunoreactive and immunoprotective determinants in individuals during naturally acquired infection will broaden our understanding of the factors involved in transmission ofand resistance to this organism and may facilitate decisions regarding public health policy and vaccine development. Acknowledgments
We gratefully appreciate the technical assistance of Jeanne Moncada. References I. Wang SP. Grayston JT. Immunologic relationship between genital TRIC, lymphogranuloma venereum, and related organisms in a new microtiter indirect immunofluorescence test. Am J Ophthalmol
1970;70:367-74. 2. Stephens RS, Tam MR, Kuo CC, Nowinski RC. Monoclonal antibodies to Chlamydia trachotnatis:antibody specificities and antigen characterization. J Immunol 1982;128: 1083-9. 3. Stephens RS. Sanchez-Pescador R. Wagar EA. Inouye C, Urdea M. Diversity of Chlamydia trachontatis major outer membrane protein genes. J Bacteriol 1987; 169:3879-85. 4. Norrby E. Biberfield G, Chiodi F, et al. Discrimination between antibodies to HIV and to related retroviruses using site-directed serology. Nature 1987;329:248-50. 5. Yuan Y. Zhang YX. Watkins N. Caldwell HD. Nucleotide and deduced amino acid sequences for the four variable domains of the major outer membrane proteins of the 15 Chlamydia trachomatis serovars. Infect Immun 1989;57: 1040-9. 6. Hanna L. Jawetz E. Briones O. et al. Antibodies to TRIC agents in tears and serum of naturally infected humans. J Infect Dis 1973; 127:
95-8. 7. Jones HM. Volpicelli M. Stephens RS. Immunoassay of polyclonal antibody responses to Chlamydia trachomatis variable segment one and variable segment two major outer membrane protein peptides. In: Bowie WR. Caldwell HD. Jones RP, et al., eds. Chlamydial infections: proceedings of the seventh international symposium on human chlamydial infections. New York: Cambridge University Press.
1990: 109-12. 8. Baehr W. Zhang YX. Joseph T, et al. Mapping antigenic domains expressed by Chlamydia trachomatis major outer membrane protein genes. Proc Nat! Acad Sci USA 1988;85:4000-4. 9. Stephens RS. Wagar EA. Schoolnik GK. High-resolution mapping of
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tions for sera obtained from a trachoma-endemic region. The preponderance of serotypes A and C in this population has been noted previously using MIF [6]. Even though our assay results and previous MIF data on the same population showed this strong C-complex representation (serovars A and C) in the trachoma patient samples, several B-complex sera among these were detected. Moreover, in preliminary evaluations with an identical experimental format of sera from patients with STDs, we could easily detect B-complex representatives (B- and E-type-specific responses; unpublished data). Thus, we believe that the SDI can be applied to both B- and C-complex serogroups. Some of the samples were not typeable by either MIF or SDI, perhaps because of sequence variability within given serovars, the existence of new, uncharacterized serovars [II ], or a low-titered response to multiple serovars. Closely related serovars, such as serovars C and J, Band Ba, G and F, or D and E, cannot be distinguished by one-way MIF assay. For example, the only difference between Band Ba is that antisera to Ba shows greater cross-reactivity with other serovars than does antisera to B. From a practical point of view, these are typically not discriminated in MIF evaluations, and this assay is often simplified by the pooling ofantigens in a target population-dependent manner. Our finding of dominant serologic responses to VS 1 may explain in part the difficulty in discriminating between closely related serovars. The VS I sequences for C and J and for G and F are identical for each pair, and Band Ba have two relatively conserved amino acid differences. Complications may also arise from humoral responses to infection simultaneously or sequentially by multiple serovars; the latter may result in an "original antigenic sin" phenomenon in which a boost with an antigen closely related to the priming antigen can elicit a strong B cell memory response to the priming antigen [12]. It was surprising that the VS2 region was not very reactive with these sera, especially since VS2 is the most sequencevariable segment. This may reflect the C-complex bias of the sample population, or this region may be involved in a more conformational epitope that would not necessarily be detected with these short linear peptides. Evidence does exist for its surface exposure in both Band C subgroups [13] (unpublished data), and serovar-specific monoclonal antibodies have been mapped to this region [8, 9]. The precise role of the anti-VSI response in chlamydial immunobiology is not yet known, and its relationship with more complicated, conformational determinants remains to be established. The SDI results in this trachoma-endemic population mimicked the results of MIF. If this concordance holds true for the serovariants that cause STDs, then the utility ofthe assay will be universal for serologic investigations of diseases of chlamydial origin. We propose that SDI is an effective alternative to MIF, and it has important practical advantages over MIF (flexibility, simplicity, low cost, objectivity, portability, and single-
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serovar-specific and common antigenic determinants of the major outer membrane protein of Chlamydia trachomatis. J Exp Med 1988; 167:817-31. 10. Schachter J, Dawson CR. Human chlamydial infections. Littleton, MA: PSG Publishing. 1978. II. Dean D. Patton M, Stephens RS. Direct sequence evaluation of the major outer membrane protein gene variant regions of Chlamydia trachomatis subtypes D'. 1', and L2'. Infect Immun 1991;59: 157982. 12. Fazekas de St Groth S. Webster RG. Disquisitions on original antigenic sin. II. Proof in lower creatures. J Exp Med 1966; 124:347-6 I.
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13. Su H. Zhang YX. Barrera O. Watkins NO. Caldwell HD. Differential effect of trypsin on infectivity of Chlamydia trachomatis:loss of'infectivity requires cleavage of major outer membrane protein variable domains II and IV. Infect Immun 1988;56:2094-100. 14. Treharne JD. Dwyer RSC, Darougar S. Jones BR. Daghfous T. Antichlamydial antibody in tears and sera and serotypes of Chlamydia trachomatis isolated from school children in southern Tunisia. Br J Ophthalmol 1978;62:509-15. 15. Taylor HR, Siler JA, Mkocha HA, et at. Longitudinal study of the microbiology of endemic trachoma. J Clin Microbiol 1991;29: 1593-5.
Pierre J. Plourde, Mark Tyndall, Elizabeth Agoki, John Ombette, Leslie A. Slaney, Lourdes J. D'Costa, Jackoniah O. Ndinya-Achola, and Francis A. Plummer
World Health Organization Centre for Research and Training on Sexually Transmitted Diseases, Department of Medical Microbiology. University of Nairobi, and Special Treatment Clinic, Nairobi City Commission, Kenya; Departments ofInternal Medicine and Medical Microbiology, University ofManitoba, Canada
Sexually transmitted diseases (STDs) have had a significant adverse effect on reproductive and child health worldwide. The control of STDs such as gonorrhea is therefore an absolute priority. Cefixime, an oral third-generation cephalosporin with in vitro activity similar to that of ceftriaxone, may be an effective candidate for the treatment of gonorrhea. The efficacy of a single oral 400-mg dose of cefixime was compared with that of a single intramuscular 250-mg dose of ceftriaxone for the treatment of Neisseria gonorrhoeae urethritis in 190 men and cervicitis in 46 women in Nairobi, Kenya. A bacteriologic cure was recorded in 100% of63 evaluatable patients treated with ceftriaxone and 118 (98%) of 121 evaluatable patients treated with cefixime. Cefixime, as a single oral dose, is an effective alternative for the treatment of uncomplicated gonococcal urethritis in men and cervicitis in women.
Sexually transmitted diseases (STDs), including gonorrhea, have had a significant adverse effect on reproductive and child health worldwide. Slowing the spread ofgonorrhea is a major public health priority at this time. Treatment of Neisseria gonorrhoeae infection has been complicated by the
Received 3 February 1992; revised 4 May 1992. Presented in part: Ninth meeting of the International Society for Sexually Transmitted Diseases Research. Banff, Canada, October 1991. This study received institutional review board approval from the University of Manitoba and the University of Nairobi. Verbal informed consent was obtained prior to enrollment into the study. Financial support: American Cyanamid Company, Lederle Laboratories; Medical Research Council ofCanada (fellowships to PJ.P. and M.T., Scientist award to EA.P.). Reprints or correspondence: Dr. Pierre J. Plourde, Department of Medical Microbiology. Basic Sciences Building. Room 544. 730 William Ave., Winnipeg. Manitoba. Canada. R3E OW3. The Journal of Infectious Diseases 1992;166:919-22 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6604-0037$01.00
emergence of chromosomally and plasmid-mediated antimicrobial-resistant strains [1]. Plasmid-mediated resistance, first reported in 1976. resulted in the emergence of penicillinase-producing N. gonorrhoeae [2]. Third-generation cephalosporins offer an excellent therapeutic choice because of their intrinsic .B-lactamase resistance. Intramuscular ceftriaxone has been established as an ideal treatment for N. gonorrhoeae [3] and is currently the recommended first-line therapy of choice [4]. However, wherever possible, it would be preferable to avoid parenteral therapy when an efficacious oral antibiotic exists. Cefixime, an orally administered third-generation cephalosporin, has favorable phamacokinetics for single-dose treatment of uncomplicated gonorrhea [5] and has high in vitro activity against antimicrobial-resistant isolates of N. gonorrhoeae [6,7]. This study was designed to determine the safety and efficacy of a single 400-mg oral dose of cefixime compared with a single 250-mg intramuscular (im) dose of ceftriaxone for
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Single-Dose Cefixime versus Single-Dose Ceftriaxone in the Treatment of Antimicrobial-Resistant Neisseria gonorrhoeae Infection
