VIRAL IMMUNOLOGY Volume 27, Number 9, 2014 ª Mary Ann Liebert, Inc. Pp. 471–477 DOI: 10.1089/vim.2014.0033
Detection of Immunoglobulin IgA and IgG Against Human Papilloma Virus Ana Katherine Gonc¸alves,1,* Paula Renata Lima Machado,2 Luanda Cana´rio de Souza,2 Ana Paula Ferreira Costa, 2 Fabrı´cia Gimenes,3 Marcia Lopes Consolaro,3 Janaina Oliveira Crispim,2 Jose Eleute´rio Jr.,4 and Paulo Ce´sar Giraldo 5,*
Abstract
The interest in human papilloma virus (HPV) seropositivity has increased considerably since HPV vaccines have become available worldwide. The aim of this study was to assess the performance of enzyme-linked immunosorbent assay (ELISA) in analyzing serum samples provided from women with and without genital DNA-HPV infection confirmed by polymerase chain reaction (PCR), for detection of specific antibodies of the isotypes IgG and IgA recognizing HPV-16 and -18, as well as virus-like particles (VLPs). From August to December 2013, 50 sexually active female patients between 18 and 35 years of age from the outpatient clinic at the university hospital were enrolled. In order to test them, positive controls were obtained from patients with HPV-induced lesions and who were DNA-HPV positive confirmed by PCR. A specific assay was used to identify antibodies to HPV VLPs by ELISA. The samples were divided into HPV positive and negative, and an ELISA detecting IgA and IgG anti-HPV-VLP was carried out. The effectiveness of ELISA and the kappa (k) index was obtained from the values entered in the receiver operating characteristic (ROC) curves for IgG and IgA. IgG-VLP-HPV-16 showed a good correlation between ELISA and PCR (k = 0.75), and IgG-VLP-HPV18 showed a very good correlation between ELISA and PCR (k = 0.84). While the IgA antibody correlation was also positive, although weaker, IgA-VLP-HPV-16 was moderate (k = 0.45) and IgA-VLP-HPV-18 good (k = 0.66). The efficacy of the assay concerning IgG was: sensitivity, specificity, and accuracy were 82.3%, 92%, and 88% to IgG-VLP-HPV-16, and 100%, 92%, and 94% to IgG-VLP-HPV-18. The assay concerning IgA was: sensitivity, specificity, and accuracy were 64.7%, 80%, and 73.8% to IgA-VLP-HPV-16, and 100%, 80%, and 84.8% to IgA-VLP-HPV-18. IgG and IgA antibodies against HPV-16 and -18 can be detected in unvaccinated individuals by using the VLP that serve as the basis for bivalent HPV vaccine. The values for ELISA assays and the values found for IgG correlate good/very good with HPV-16/18 detected by PCR. Introduction
H
uman papilloma virus (HPV) is one of the most common sexually transmitted infections worldwide. Up to 70% of sexually active women will contract HPV during their life (2). HPV is related to six types of carcinomas: cervix, penis, vulva, vagina, anus, and oropharynx. Invasive cervical cancer (ICC) has long been a significant complication of genital HPV infection, and is the second most common cancer affecting women worldwide. There are approximately 500,000 new cases of ICC reported each year, and more than
270,000 cases per year are fatal (2,22,27). Fifteen oncogenic HPV types have been identified. However, serotypes 16 and 18 are the most commonly found (9,21). HPV has been well established as a necessary cause of ICC (4). Persistent HPV-16 and -18 genital infections are a common denominator in the progression of cervical intraepithelial neoplasia and have been found to increase the risk of subsequent development of invasive ICC (5,8). Around the world, 70% of ICC cases are caused by HPV-16 or -18, HPV-16 being the most commonly detected in 55% of cases, followed by HPV-18 in 15% of cases (28,33).
Departments of 1Obstetrics and Gynecology and 2Toxicology and Clinical Analysis, Universidade Federal do Rio Grande do Norte, Natal-RN, Brazil. 3 Section of Clinical Cytology, Department of Clinical Analysis and Biomedicine, Universidade Estadual de Maringa´, Maringa-PR, Brazil. 4 Department of Obstetrics and Gynecology, Universidade Federal do Ceara´, Fortaleza-CE, Brazil. 5 Department of Obstetrics and Gynecology, Universidade Estadual de Campinas, Campinas-SP, Brazil. * These authors contributed equally to this work.
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Since chronic HPV infection causes cervical lesions that may progress to more advanced stages of the ICC, HPV antibody positivity could become a biomarker for predicting cancer risk in its reflection of persistent long-term disease. A past study reported that HPV-16-VLP-specific immunoglobulin G (IgG) was not only detected in women who had a persistent HPV infection, but also in women with cervical lesions that eventually progressed to cancer (26). However, the most asymptomatic HPV infections clear without treatment, and a substantial proportion of low-grade lesions regress without treatment. It is only when the virus remains for more than a year that the infected patient risks developing ICC. The medical community has been battling to increase host immunity by means of vaccination and preventive therapy for some time (1). HPV vaccination induces production of neutralizing antibodies that protect against infection and disease. Therefore, type-specific antibody detection plays a key role in determining efficacy, quality, potency, and duration of immune responses to HPV vaccination and eventual correlates of protection. Vaccination of patients with preventative viral-like particles (VLP) seems to increase HPV antibody titers significantly. In contrast, natural HPV infections have been found to result from low antibodies (4,18). The HPV vaccine stimulates a humoral immune response by exposing the system to VLPs, noninfectious structures that simulate HPV infection. The oncogenic types chosen for both vaccines were those responsible for the majority of ICC cases worldwide: HPV-16 and -18 (4,18). HPV immunity is not completely understood, and there is currently no standard commercially available serologic test that detects HPV-16 and -18 antibodies. Clinical applications of assays are difficult due the low titers of anti-VLPs and HPV caused by the immune seclusion of the cervix, as well as the disease’s lack of viremic phase. Thus, the latter is the principal barrier to the development of a clinically relevant assay for HPV antibody screening. Nevertheless, HPV serum antibodies could supply a cumulative measure of viral exposure that may be significant in studies of vaccinated and unvaccinated populations. The objective of this study was to determine the performance of enzyme-linked immunosorbent assay (ELISA) in detecting antibodies for HPV-VLPs. IgA and IgG responses were documented for both HPV-16/18, and the sensitivity, specificity, and reproducibility of each was recorded. Methods Patients
From August 2013 to December 2013, 50 sexually active women, between 18 and 35 years of age from the outpatient clinic at university hospital were invited to participate in the study. Positive controls were obtained from patients with genital HPV-induced lesions and who were DNA-HPV positive confirmed by polymerase chain reaction (PCR), while negative controls were obtained from DNA-HPV negative women confirmed by PCR. In the positive controls, the seroconversion was detected more than 18 months after the detection of HPV-16/18 DNA to avoid samples from women with transient HPV-DNA infection associated with a failure to seroconvert (3).
Genital samples for PCR and routine Pap smear and colposcopic examinations were performed to confirm or exclude the possibility of subclinical HPV lesions. Women who were pregnant, menopausal, using immunosuppressive medications or antibiotics, and presenting oral infections and/or inflammations or degenerative diseases were excluded. Samples
Initially, cervical samples were collected to confirm or exclude HPV infection and identify the type of HPV for PCR. Once HPV infection had been confirmed or excluded, peripheral blood was collected by venipuncture. The aliquots of blood were collected into tubes containing separating gel, and were coagulated at room temperature and centrifuged at 3,000 g to obtain the serum. IgA/IgG levels were then measured. Positive controls were obtained from HPV-16/18 seropositive patients (NICI, NICII, NICIII, carcinoma, and HPV), while negative controls were obtained from seronegative women confirmed by PCR. All participants provided written informed consent. The project protocol was reviewed and approved by the Ethical Committee at of The Universidade Estadual de Campinas (1034/2011 CEPUNICAMP). Molecular detection of HPV Extraction of DNA-HPV. Exfoliated cervicovaginal cells were obtained by lavage for HPV determination and typing by PCR. Samples of cervical mucosa were collected with endocervical cytobrushes attached to tubes containing a preservative solution (DNA collection device kit; DIGENE). The samples were then stored at a temperature of - 70C. At the time of extraction, the samples were thawed and vortexed for 30 sec. All content from the collection tube was transferred to microtubes of 1.5 mL previously identified with the corresponding sample number. DNA extractions were performed following the protocol of the AxyPrep Body Fluid Viral DNA/RNA Miniprep kit (Axygen Biosciences) using the V-L, V-N, W1A, W2, TE buffers according to the manufacturer’s instruction. PCR screening for HPV. DNA-HPV was amplified with GP5 + and GP6 + primers as described previously (15). A final volume of 25 lL was used in the amplification reaction to detect DNA-HPV: this consisted of 12.5 lL AmpliTaq Gold 360 Master Mix (Applied Biosystems), 20 pmol of each primer, 5.5 lL of deionized water, and 5 lL of extracted DNA. The amplification reaction was carried out under the following conditions: (a) preheating at 94C for 4 min; (b) 40 cycles of 94C for 1 min, 40C for 2 min, and 72C for 1 min; and (c) 72C for 4 min. The gel was stained with SYBR Safe DNA Gel Stain (Life Technologies) and a molecular weight marker of 100 bp was used for all electrophoretic races (Fermentas). The 150 bp amplified fragments of DNA were viewed using a transilluminator with UV light and then photographed in the photodocumentation system. The quality of the amplified DNA was tested by amplification of the human beta-globin gene. The primers used were GL1 (5¢-TGA GTC CTT TGG GGA TCT GTC CA-3¢)
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and GR2 (5¢-TGA AGT TCT CAG GAT CCA CGT GC-3¢), which amplify segments of 185 bp. The positive controls were DNA extracted of SiHa and HeLa cells infected with HPV-16 and -18 respectively. As a negative control, all PCR reagents were added to a 0.2 mL microtube without DNA. PCR for HPV-16/18. The E7 genes of HPV-16/-18 were amplified using the following primers: 5¢-ATT AAA TGA CAG CTC AGA GGA-3¢ and 5¢-GCT TTG TAC GCA CAA CCG AAG C-3¢ (7) which amplify a segment of 133 pb in the region of the HPV-16 E7 gene, and 5¢-AAG AAA ACG ATG AAA TAG ATG GA-3¢ and 5¢-GGC TTC ACA CTT ACA ACA CA-3¢, which amplify a segment of 104 pb in the region of the HPV-18 E7 gene. A final volume of 25 lL was used in the amplification reaction, which consisted of 12.5 lL AmpliTaq Gold 360 Master Mix (Applied Biosystems), 20 pmol of each primer (the two primers for each respective HPV type), 5.5 lL of deionized water, and 5 lL of extracted DNA. The amplification reaction was performed under the following conditions: (a) preheating at 94C for 10 min; (b) 45 cycles of 94C for 1 min, 55C for 1 min, and 72C for 2 min; (c) 72C for 10 min. ELISA for detection of IgA and IgG anti-HPV-VLP. Details of the antigen preparation have been described previously (28). Initially, a plate of 96 wells was sensitized with 50 lL of antigen (vaccine for HPV-16 and HPV-18) diluted in carbonate-bicarbonate buffer (Sigma-Aldrich) at a concentration of 10 lg/mL and incubated at 4C overnight. The plate was then washed three times with PBS-Tween 0.05% and blocked with 100 lL of phosphate-buffered saline (PBS; pH = 7.4), which contained 3% nonfat milk (SigmaAldrich). It was then incubated for 2 h at room temperature and finally washed three times with PBS-Tween 0.05%. Serum samples were then diluted 1:100 in PBS containing 3% nonfat milk, and 50 lL of the resulting mixture was added to each well. Following this, the samples were incubated for 2 h at 37C, and the plate was washed three times with PBS-Tween 0.05%. Next, secondary antibody (peroxidase-labeled anti-human IgG or anti-human IgA; Sigma-Aldrich) was diluted 1:10,000 in PBS containing 3% nonfat milk, and 50 lL was added to the wells and incubated for 1 h at 37C.
The plate was washed five times with PBS-Tween 0.05% and then 50 lL of substrate TMB (3,3¢,5,5¢-Tetramethylbenzidine Liquid Substrate System; Sigma-Aldrich) was added to the wells before incubating for 30 min at room temperature. The reaction was stopped with 50 lL of 1 N sulfuric acid, and the absorbance (optical density) of each well was read using an ELISA reader at 450 nm with a reference filter of 630 nm. Statistical analysis
The first 25 positive HPV samples and the first 25 negative HPV samples were used for the construction of the receiver operating characteristic (ROC) curve. The cutoff was established from the ROC curve calculated using the BioEstat 5.3 program. Agreement of PCR and detection of IgG/IgA anti-HPV was tested by the weighted kappa statistic (28,32). Results
The ELISA assay for HPV-VLP (HPV-VLPs ELISA) was carried out using 25 seronegative and 25 seropositive HPV samples. It was observed that approximately 30% of the seronegative samples reacted when the antigen was not present. To improve the specificity of the assay, the concentrations of both antigen and primary antibody were gradually reduced. Following extensive tests, the optimal antigen dilution was found to be 10 lg/mL of vaccine diluted in a carbonate bicarbonate buffer. Changing the temperature to 4C led to a significant improvement in specificity. Under these conditions, subsequent tests were performed. Agreement between ELISA and PCR was tested using the weighted kappa coefficient, using the categories proposed by Landis and Koch (19). In this study, the correlation between ELISA for IgG-VLP-HPV-16 and PCR was found to be good (weighted kappa coefficient = 0.75 [95% CI 0.546–0.955]). The correlation between ELISA for IgG-VLP-HPV-18 and PCR was found to be very good (weighted kappa coefficient = 0.75 [95% CI 0.546–0.955]). Concerning IgA, the correlation between ELISA for IgA-VLP-HPV-16 and PCR was found to be moderate (weighted kappa coefficient = 0.451 [95% CI 0.176–0.727]). The correlation between ELISA for IgA-VLP-HPV-18 and PCR was found to be good (weighted kappa coefficient = 0.660 [95% CI 0.401–0.918]).
Table 1. Agreement Between ELISA for IgG/IgA-VLP-HPV and PCR Anti-HPV IgG Samples HPV-16 DNA positive HPV-18 DNA positive HPV-16/-18 DNA negative (control)
Anti-HPV IgA
Positive
Negative
p
Positive
Negative
p
14 8 2
3 0 23
< 0.0001 < 0.0001
11 8 5
6 0 20
0.0084 < 0.0001
Statistical significance obtained using the Fisher’s exact test. HPV IgG. HPV-16 positive vs. control. Sensitivity 82.3% (56.6–96.2). Specificity 92% (73.9–99). PPV 87.5% (61.6–98.4). NPV 88.5% (69.8–97.5). Accuracy 88%. Anti-HPV IgG. HPV-18 positive vs. control. Sensitivity 100% (63.1–100). Specificity 92% (74–99). PPV 80% (44.4–97.5). NPV 100% (85.2–100). Accuracy 94%. Anti-HPV IgA. HPV-16 positive vs. control. Sensitivity 64.7% (38.3–85.8). Specificity 80% (59.3–93.2). PPV 68.7% (41.3–89). NPV 76.9% (56.3–91). Accuracy 73.8%. Anti-HPV IgA. HPV-18 positive vs. control. Sensitivity 100% (63.1–100). Specificity 80% (59.3–93.2). PPV 61.5 (31.6–86.1). NPV 100% (83.2–100). Accuracy 84.8%. ELISA, enzyme-linked immunosorbent assay; HPV, human papilloma virus; PCR, polymerase chain reaction; IgG, immunoglobulin G; PPV, positive predictive value; NPV, negative predictive value.
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FIG. 1. Received operating characteristic (ROC) curve obtained from analysis of the enzyme-linked immunosorbent assay (ELISA) results for the detection of immunoglobulin G (IgG) or IgA anti-HPV with polymerase chain reaction (PCR) as the comparison method. (A) IgG anti-HPV-16 versus control. (B) IgG anti-HPV-18 versus control. (C) IgA anti-HPV-16 versus control. (D) IgA anti-HPV-18 versus control. Regarding IgG and IgA, serum samples were used to compare the data of sensitivity, specificity, and efficacy of the assay (Table 1). The performances of both tests were compared using the ROC curve (Fig. 1). The detection of antibodies against HPV-VLPs was strongly correlated with the presence of DNA-HPV. The effectiveness of the ELISA assay for IgG was observed as:
Anti-HPV IgG. HPV-16 positive versus control: sensitivity 82%, specificity 92%, positive predictive value (PPV) 87%, negative predictive value (NPV) 88%, accuracy 88%. Anti-HPV IgG. HPV-18 positive versus control: sensitivity 100%, specificity 92%, PPV 80%, NPV 85%, and accuracy 94%. The effectiveness of the ELISA assay for IgA was observed as:
Anti-HPV IgA. HPV-16 positive versus control: sensitivity 64%, specificity 80%, PPV 68%, NPV 76%, and accuracy 73%. Anti-HPV IgA. HPV-18 positive versus control: sensitivity 100%, specificity 80%, PPV 61%, NPV 100%, and accuracy 84%. Discussion
The interest in HPV seropositivity has increased considerably since HPV vaccines have become available world-
wide. Serological assays are used to screen volunteers for vaccine trials as to previous exposure to HPV, and to evaluate seroconversion in vaccines. In post-vaccination studies, monitoring vaccination performance (immunogenicity) and vaccination coverage, and surveillance of correlates of protection in vaccinated cohorts are potential areas for using antibody-based measurements in the future (25). Serological assay also could become an interesting alternative method for detecting HPV infection, since it is more easily manipulated and does not require special equipment. It could predict past and present HPV infection by systematically detecting immune response against HPV infection. However, a reliable assay of this kind still does not exist because of the difficulty of obtaining viral antigens by traditional purification methods (5). In our study, similar to others (3,25,29), an extensive number of tests were necessary to find the optimal antigen dilution, which was found to be 10 lg/mL of vaccine diluted in carbonate bicarbonate buffer. The adoption of a temperature of 4C brought on a significant improvement in specificity. In others words, this reduced false positives in the negative samples and increased intra-assay reproducibility. This study, like others (3), also came across another obstacle: the low ranges of optical densities seen mainly in the IgA assay. Studies have demonstrated that specific IgA for HPV VLPs correlates with specific IgG or the DNA-HPV detection (23). However, such studies have concentrated only on the serum IgG responses to HPV-16 and -18 (12,16,24).
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Additionally, it is important to recognize that IgA is one of the most abundant isotypes in serum and plays an important role in the protection against pathological agents mainly at genital mucosal sites (10,11,23). Other studies (15,16,23,24) have similarly observed that IgG and IgA were detected much more frequently in positive samples than in negative samples. Our results also observed that the detection of antibodies against HPV was strongly correlated with the presence of DNA-HPV, confirming the association between serum immunoglobulin responses and DNA-HPV. Our results also conclude that the accuracy of the test was higher for IgG (88/93%), 16/18HPV, respectively than IgA (73%/93%), 16/18HPV, respectively. This may be explained by the fact that IgA remains in the serum for a shorter period of time (16,23). The literature reports that IgG seroconversion against HPV occurs within 6–12 months after infection and that IgG response remains high over a long period of time (12,23). Thus, the duration of IgA in sera and cervical secretions is shorter than the duration of serum IgG (23). Earlier studies have demonstrated that a substantial proportion of women with cervical HPV infection do not seroconvert (3,14,23,31). Carter et al. (3) reported that approximately 60% of their subjects seroconverted within 18 months. These data suggest that development of antibodies at a detectable level after a natural infection can be a slow process, and it does not necessarily occur in every woman. Several factors may explain this event, such as antibodies to HPV circulate at a low titer, low levels of antibodies need to be boosted over time so a detectable seroconversion may take time, and differences in the host genetic background or immune status could also explain why some individuals failed to seroconvert. Carter et al. (3) also reported that individuals who were repeatedly HPV-DNA negative after seroconversion tended not to have persistent IgG antibodies, and this phenomenon was true for antibody responses against HPV-16, HPV-18, and HPV-6 (3). On the other hand, some studies have shown that failure to seroconvert was associated with having had only one HPV-DNA-positive visit for HPV-16. Seroconversion to HPV-18 was also detected less frequently among women who had only one HPV-18 DNA-positive visit than among women with multiple HPV-18 DNA-positive visits (3,6,34). The detection of HPV-16 antibodies is also associated with HPV-16 DNA persistence (3,6,34). De Gruijl et al. (6) found that persistent HPV-16 DNA and HPV-16 serum antibodies were present in all women who developed severe dysplasia in their study. In our study, the seroconversion was detected in patients with persistent HPV infection (HPV injury) and more than 18 months after the detection of HPV-16/18 DNA to avoid samples from women with transient HPV-DNA infection associated with a failure to seroconvert. We have shown herein that the proportion of women who seroconverted (IgG) was more than 80% after 18 months of DNA detection for HPV-16 (sensitivity 82%) and HPV-18 (sensitivity 100%). The better results of our study also could be explained by the significant improvement the sensitivity of antibody assays and evolution of the method during the last 10 years. In order to establish standards for HPV antibody detection, the World Health Organization has coordinated an interna-
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tional collaborative study (8). The results of this study suggest that the establishment of international standard reagents for antibodies to HPV would promote the comparison of HPV antibody measurements between assays and laboratories worldwide. HPV standard reagents for serology will be essential to improve the sensitivity of antibody assays, the consistency of results worldwide, as well as improving interlaboratory and interassay variability, as recommended in comparative studies (29). Besides this, standardization would increase assay performance, allow valid comparison of results between laboratories, and facilitate monitoring of vaccine effectiveness (13,14,23,25). Until now, serology has played no role in the diagnosis of HPV infections, despite serological procedures for the disclosure to and infection with viral pathogens being the usual mode of documentation of viral infection (3,5). There is no classical ‘‘gold standard’’ for measuring HPV serological responses, and different assays may be preferable depending on the questions being asked. The presence of HPV antibodies in the bloodstream of individuals is mostly a marker for past infections, not necessarily correlating to present infection or disease. HPV seropositivity may result from a prior viral infection that has been efficiently cleared, as well from an asymptomatic infection. Consequently, serological assays have not proven their clinical diagnostic value thus far (25). Nevertheless, HPV serology has proven valuable in epidemiological studies based on seropositivity for HPV to understand the natural history of infections and cancer. Importantly, antibody-based immune responses to HPV have contributed notably to understanding the protection provided by neutralizing antibodies, and have allowed for important advancements in the development of a vaccine (13,25). In the context of vaccination, serological assays could be used to screen subjects for suitability and evaluate seroconversion in the vaccine. Moreover, the use of international standardization will guarantee a universal protocol (17,20,25,29,30). In conclusion, for assessment of cumulative exposure to genital HPV infection prior to vaccination, the preferred assay to use may be the VLP ELISA, since it is the most commonly used as well as the easiest to find and least expensive. Thus, ELISA may be useful as a complementary diagnostic tool to the well-established PCR method. Besides this, a standardization of ELISA could be used to monitor the performance of HPV vaccines around the world. However, the availability of high-quality VLPs may be a limiting factor. Some form of quality control for both the vaccine and VLPs needs to be established such as monitoring vaccine performance in laboratories: this would be particularly useful in the attempt to study immune correlates of protection. Acknowledgments
Financially supported by the FAPESP (Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo). Process number: 2011/19960-9. Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to: Dr. Ana Katherine Gonc¸alves Department of Obstetrics and Gynecology Universidade Federal do Rio Grande do Norte Rua General Cordeiro de Farias S/N Petropo´lis Natal–RN 59012-570 Brazil E-mail: [email protected]
Detection of Immunoglobulin IgA and IgG Against Human Papilloma Virus Ana Katherine Gonc¸alves,1,* Paula Renata Lima Machado,2 Luanda Cana´rio de Souza,2 Ana Paula Ferreira Costa, 2 Fabrı´cia Gimenes,3 Marcia Lopes Consolaro,3 Janaina Oliveira Crispim,2 Jose Eleute´rio Jr.,4 and Paulo Ce´sar Giraldo 5,*
Abstract
The interest in human papilloma virus (HPV) seropositivity has increased considerably since HPV vaccines have become available worldwide. The aim of this study was to assess the performance of enzyme-linked immunosorbent assay (ELISA) in analyzing serum samples provided from women with and without genital DNA-HPV infection confirmed by polymerase chain reaction (PCR), for detection of specific antibodies of the isotypes IgG and IgA recognizing HPV-16 and -18, as well as virus-like particles (VLPs). From August to December 2013, 50 sexually active female patients between 18 and 35 years of age from the outpatient clinic at the university hospital were enrolled. In order to test them, positive controls were obtained from patients with HPV-induced lesions and who were DNA-HPV positive confirmed by PCR. A specific assay was used to identify antibodies to HPV VLPs by ELISA. The samples were divided into HPV positive and negative, and an ELISA detecting IgA and IgG anti-HPV-VLP was carried out. The effectiveness of ELISA and the kappa (k) index was obtained from the values entered in the receiver operating characteristic (ROC) curves for IgG and IgA. IgG-VLP-HPV-16 showed a good correlation between ELISA and PCR (k = 0.75), and IgG-VLP-HPV18 showed a very good correlation between ELISA and PCR (k = 0.84). While the IgA antibody correlation was also positive, although weaker, IgA-VLP-HPV-16 was moderate (k = 0.45) and IgA-VLP-HPV-18 good (k = 0.66). The efficacy of the assay concerning IgG was: sensitivity, specificity, and accuracy were 82.3%, 92%, and 88% to IgG-VLP-HPV-16, and 100%, 92%, and 94% to IgG-VLP-HPV-18. The assay concerning IgA was: sensitivity, specificity, and accuracy were 64.7%, 80%, and 73.8% to IgA-VLP-HPV-16, and 100%, 80%, and 84.8% to IgA-VLP-HPV-18. IgG and IgA antibodies against HPV-16 and -18 can be detected in unvaccinated individuals by using the VLP that serve as the basis for bivalent HPV vaccine. The values for ELISA assays and the values found for IgG correlate good/very good with HPV-16/18 detected by PCR. Introduction
H
uman papilloma virus (HPV) is one of the most common sexually transmitted infections worldwide. Up to 70% of sexually active women will contract HPV during their life (2). HPV is related to six types of carcinomas: cervix, penis, vulva, vagina, anus, and oropharynx. Invasive cervical cancer (ICC) has long been a significant complication of genital HPV infection, and is the second most common cancer affecting women worldwide. There are approximately 500,000 new cases of ICC reported each year, and more than
270,000 cases per year are fatal (2,22,27). Fifteen oncogenic HPV types have been identified. However, serotypes 16 and 18 are the most commonly found (9,21). HPV has been well established as a necessary cause of ICC (4). Persistent HPV-16 and -18 genital infections are a common denominator in the progression of cervical intraepithelial neoplasia and have been found to increase the risk of subsequent development of invasive ICC (5,8). Around the world, 70% of ICC cases are caused by HPV-16 or -18, HPV-16 being the most commonly detected in 55% of cases, followed by HPV-18 in 15% of cases (28,33).
Departments of 1Obstetrics and Gynecology and 2Toxicology and Clinical Analysis, Universidade Federal do Rio Grande do Norte, Natal-RN, Brazil. 3 Section of Clinical Cytology, Department of Clinical Analysis and Biomedicine, Universidade Estadual de Maringa´, Maringa-PR, Brazil. 4 Department of Obstetrics and Gynecology, Universidade Federal do Ceara´, Fortaleza-CE, Brazil. 5 Department of Obstetrics and Gynecology, Universidade Estadual de Campinas, Campinas-SP, Brazil. * These authors contributed equally to this work.
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Since chronic HPV infection causes cervical lesions that may progress to more advanced stages of the ICC, HPV antibody positivity could become a biomarker for predicting cancer risk in its reflection of persistent long-term disease. A past study reported that HPV-16-VLP-specific immunoglobulin G (IgG) was not only detected in women who had a persistent HPV infection, but also in women with cervical lesions that eventually progressed to cancer (26). However, the most asymptomatic HPV infections clear without treatment, and a substantial proportion of low-grade lesions regress without treatment. It is only when the virus remains for more than a year that the infected patient risks developing ICC. The medical community has been battling to increase host immunity by means of vaccination and preventive therapy for some time (1). HPV vaccination induces production of neutralizing antibodies that protect against infection and disease. Therefore, type-specific antibody detection plays a key role in determining efficacy, quality, potency, and duration of immune responses to HPV vaccination and eventual correlates of protection. Vaccination of patients with preventative viral-like particles (VLP) seems to increase HPV antibody titers significantly. In contrast, natural HPV infections have been found to result from low antibodies (4,18). The HPV vaccine stimulates a humoral immune response by exposing the system to VLPs, noninfectious structures that simulate HPV infection. The oncogenic types chosen for both vaccines were those responsible for the majority of ICC cases worldwide: HPV-16 and -18 (4,18). HPV immunity is not completely understood, and there is currently no standard commercially available serologic test that detects HPV-16 and -18 antibodies. Clinical applications of assays are difficult due the low titers of anti-VLPs and HPV caused by the immune seclusion of the cervix, as well as the disease’s lack of viremic phase. Thus, the latter is the principal barrier to the development of a clinically relevant assay for HPV antibody screening. Nevertheless, HPV serum antibodies could supply a cumulative measure of viral exposure that may be significant in studies of vaccinated and unvaccinated populations. The objective of this study was to determine the performance of enzyme-linked immunosorbent assay (ELISA) in detecting antibodies for HPV-VLPs. IgA and IgG responses were documented for both HPV-16/18, and the sensitivity, specificity, and reproducibility of each was recorded. Methods Patients
From August 2013 to December 2013, 50 sexually active women, between 18 and 35 years of age from the outpatient clinic at university hospital were invited to participate in the study. Positive controls were obtained from patients with genital HPV-induced lesions and who were DNA-HPV positive confirmed by polymerase chain reaction (PCR), while negative controls were obtained from DNA-HPV negative women confirmed by PCR. In the positive controls, the seroconversion was detected more than 18 months after the detection of HPV-16/18 DNA to avoid samples from women with transient HPV-DNA infection associated with a failure to seroconvert (3).
Genital samples for PCR and routine Pap smear and colposcopic examinations were performed to confirm or exclude the possibility of subclinical HPV lesions. Women who were pregnant, menopausal, using immunosuppressive medications or antibiotics, and presenting oral infections and/or inflammations or degenerative diseases were excluded. Samples
Initially, cervical samples were collected to confirm or exclude HPV infection and identify the type of HPV for PCR. Once HPV infection had been confirmed or excluded, peripheral blood was collected by venipuncture. The aliquots of blood were collected into tubes containing separating gel, and were coagulated at room temperature and centrifuged at 3,000 g to obtain the serum. IgA/IgG levels were then measured. Positive controls were obtained from HPV-16/18 seropositive patients (NICI, NICII, NICIII, carcinoma, and HPV), while negative controls were obtained from seronegative women confirmed by PCR. All participants provided written informed consent. The project protocol was reviewed and approved by the Ethical Committee at of The Universidade Estadual de Campinas (1034/2011 CEPUNICAMP). Molecular detection of HPV Extraction of DNA-HPV. Exfoliated cervicovaginal cells were obtained by lavage for HPV determination and typing by PCR. Samples of cervical mucosa were collected with endocervical cytobrushes attached to tubes containing a preservative solution (DNA collection device kit; DIGENE). The samples were then stored at a temperature of - 70C. At the time of extraction, the samples were thawed and vortexed for 30 sec. All content from the collection tube was transferred to microtubes of 1.5 mL previously identified with the corresponding sample number. DNA extractions were performed following the protocol of the AxyPrep Body Fluid Viral DNA/RNA Miniprep kit (Axygen Biosciences) using the V-L, V-N, W1A, W2, TE buffers according to the manufacturer’s instruction. PCR screening for HPV. DNA-HPV was amplified with GP5 + and GP6 + primers as described previously (15). A final volume of 25 lL was used in the amplification reaction to detect DNA-HPV: this consisted of 12.5 lL AmpliTaq Gold 360 Master Mix (Applied Biosystems), 20 pmol of each primer, 5.5 lL of deionized water, and 5 lL of extracted DNA. The amplification reaction was carried out under the following conditions: (a) preheating at 94C for 4 min; (b) 40 cycles of 94C for 1 min, 40C for 2 min, and 72C for 1 min; and (c) 72C for 4 min. The gel was stained with SYBR Safe DNA Gel Stain (Life Technologies) and a molecular weight marker of 100 bp was used for all electrophoretic races (Fermentas). The 150 bp amplified fragments of DNA were viewed using a transilluminator with UV light and then photographed in the photodocumentation system. The quality of the amplified DNA was tested by amplification of the human beta-globin gene. The primers used were GL1 (5¢-TGA GTC CTT TGG GGA TCT GTC CA-3¢)
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and GR2 (5¢-TGA AGT TCT CAG GAT CCA CGT GC-3¢), which amplify segments of 185 bp. The positive controls were DNA extracted of SiHa and HeLa cells infected with HPV-16 and -18 respectively. As a negative control, all PCR reagents were added to a 0.2 mL microtube without DNA. PCR for HPV-16/18. The E7 genes of HPV-16/-18 were amplified using the following primers: 5¢-ATT AAA TGA CAG CTC AGA GGA-3¢ and 5¢-GCT TTG TAC GCA CAA CCG AAG C-3¢ (7) which amplify a segment of 133 pb in the region of the HPV-16 E7 gene, and 5¢-AAG AAA ACG ATG AAA TAG ATG GA-3¢ and 5¢-GGC TTC ACA CTT ACA ACA CA-3¢, which amplify a segment of 104 pb in the region of the HPV-18 E7 gene. A final volume of 25 lL was used in the amplification reaction, which consisted of 12.5 lL AmpliTaq Gold 360 Master Mix (Applied Biosystems), 20 pmol of each primer (the two primers for each respective HPV type), 5.5 lL of deionized water, and 5 lL of extracted DNA. The amplification reaction was performed under the following conditions: (a) preheating at 94C for 10 min; (b) 45 cycles of 94C for 1 min, 55C for 1 min, and 72C for 2 min; (c) 72C for 10 min. ELISA for detection of IgA and IgG anti-HPV-VLP. Details of the antigen preparation have been described previously (28). Initially, a plate of 96 wells was sensitized with 50 lL of antigen (vaccine for HPV-16 and HPV-18) diluted in carbonate-bicarbonate buffer (Sigma-Aldrich) at a concentration of 10 lg/mL and incubated at 4C overnight. The plate was then washed three times with PBS-Tween 0.05% and blocked with 100 lL of phosphate-buffered saline (PBS; pH = 7.4), which contained 3% nonfat milk (SigmaAldrich). It was then incubated for 2 h at room temperature and finally washed three times with PBS-Tween 0.05%. Serum samples were then diluted 1:100 in PBS containing 3% nonfat milk, and 50 lL of the resulting mixture was added to each well. Following this, the samples were incubated for 2 h at 37C, and the plate was washed three times with PBS-Tween 0.05%. Next, secondary antibody (peroxidase-labeled anti-human IgG or anti-human IgA; Sigma-Aldrich) was diluted 1:10,000 in PBS containing 3% nonfat milk, and 50 lL was added to the wells and incubated for 1 h at 37C.
The plate was washed five times with PBS-Tween 0.05% and then 50 lL of substrate TMB (3,3¢,5,5¢-Tetramethylbenzidine Liquid Substrate System; Sigma-Aldrich) was added to the wells before incubating for 30 min at room temperature. The reaction was stopped with 50 lL of 1 N sulfuric acid, and the absorbance (optical density) of each well was read using an ELISA reader at 450 nm with a reference filter of 630 nm. Statistical analysis
The first 25 positive HPV samples and the first 25 negative HPV samples were used for the construction of the receiver operating characteristic (ROC) curve. The cutoff was established from the ROC curve calculated using the BioEstat 5.3 program. Agreement of PCR and detection of IgG/IgA anti-HPV was tested by the weighted kappa statistic (28,32). Results
The ELISA assay for HPV-VLP (HPV-VLPs ELISA) was carried out using 25 seronegative and 25 seropositive HPV samples. It was observed that approximately 30% of the seronegative samples reacted when the antigen was not present. To improve the specificity of the assay, the concentrations of both antigen and primary antibody were gradually reduced. Following extensive tests, the optimal antigen dilution was found to be 10 lg/mL of vaccine diluted in a carbonate bicarbonate buffer. Changing the temperature to 4C led to a significant improvement in specificity. Under these conditions, subsequent tests were performed. Agreement between ELISA and PCR was tested using the weighted kappa coefficient, using the categories proposed by Landis and Koch (19). In this study, the correlation between ELISA for IgG-VLP-HPV-16 and PCR was found to be good (weighted kappa coefficient = 0.75 [95% CI 0.546–0.955]). The correlation between ELISA for IgG-VLP-HPV-18 and PCR was found to be very good (weighted kappa coefficient = 0.75 [95% CI 0.546–0.955]). Concerning IgA, the correlation between ELISA for IgA-VLP-HPV-16 and PCR was found to be moderate (weighted kappa coefficient = 0.451 [95% CI 0.176–0.727]). The correlation between ELISA for IgA-VLP-HPV-18 and PCR was found to be good (weighted kappa coefficient = 0.660 [95% CI 0.401–0.918]).
Table 1. Agreement Between ELISA for IgG/IgA-VLP-HPV and PCR Anti-HPV IgG Samples HPV-16 DNA positive HPV-18 DNA positive HPV-16/-18 DNA negative (control)
Anti-HPV IgA
Positive
Negative
p
Positive
Negative
p
14 8 2
3 0 23
< 0.0001 < 0.0001
11 8 5
6 0 20
0.0084 < 0.0001
Statistical significance obtained using the Fisher’s exact test. HPV IgG. HPV-16 positive vs. control. Sensitivity 82.3% (56.6–96.2). Specificity 92% (73.9–99). PPV 87.5% (61.6–98.4). NPV 88.5% (69.8–97.5). Accuracy 88%. Anti-HPV IgG. HPV-18 positive vs. control. Sensitivity 100% (63.1–100). Specificity 92% (74–99). PPV 80% (44.4–97.5). NPV 100% (85.2–100). Accuracy 94%. Anti-HPV IgA. HPV-16 positive vs. control. Sensitivity 64.7% (38.3–85.8). Specificity 80% (59.3–93.2). PPV 68.7% (41.3–89). NPV 76.9% (56.3–91). Accuracy 73.8%. Anti-HPV IgA. HPV-18 positive vs. control. Sensitivity 100% (63.1–100). Specificity 80% (59.3–93.2). PPV 61.5 (31.6–86.1). NPV 100% (83.2–100). Accuracy 84.8%. ELISA, enzyme-linked immunosorbent assay; HPV, human papilloma virus; PCR, polymerase chain reaction; IgG, immunoglobulin G; PPV, positive predictive value; NPV, negative predictive value.
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FIG. 1. Received operating characteristic (ROC) curve obtained from analysis of the enzyme-linked immunosorbent assay (ELISA) results for the detection of immunoglobulin G (IgG) or IgA anti-HPV with polymerase chain reaction (PCR) as the comparison method. (A) IgG anti-HPV-16 versus control. (B) IgG anti-HPV-18 versus control. (C) IgA anti-HPV-16 versus control. (D) IgA anti-HPV-18 versus control. Regarding IgG and IgA, serum samples were used to compare the data of sensitivity, specificity, and efficacy of the assay (Table 1). The performances of both tests were compared using the ROC curve (Fig. 1). The detection of antibodies against HPV-VLPs was strongly correlated with the presence of DNA-HPV. The effectiveness of the ELISA assay for IgG was observed as:
Anti-HPV IgG. HPV-16 positive versus control: sensitivity 82%, specificity 92%, positive predictive value (PPV) 87%, negative predictive value (NPV) 88%, accuracy 88%. Anti-HPV IgG. HPV-18 positive versus control: sensitivity 100%, specificity 92%, PPV 80%, NPV 85%, and accuracy 94%. The effectiveness of the ELISA assay for IgA was observed as:
Anti-HPV IgA. HPV-16 positive versus control: sensitivity 64%, specificity 80%, PPV 68%, NPV 76%, and accuracy 73%. Anti-HPV IgA. HPV-18 positive versus control: sensitivity 100%, specificity 80%, PPV 61%, NPV 100%, and accuracy 84%. Discussion
The interest in HPV seropositivity has increased considerably since HPV vaccines have become available world-
wide. Serological assays are used to screen volunteers for vaccine trials as to previous exposure to HPV, and to evaluate seroconversion in vaccines. In post-vaccination studies, monitoring vaccination performance (immunogenicity) and vaccination coverage, and surveillance of correlates of protection in vaccinated cohorts are potential areas for using antibody-based measurements in the future (25). Serological assay also could become an interesting alternative method for detecting HPV infection, since it is more easily manipulated and does not require special equipment. It could predict past and present HPV infection by systematically detecting immune response against HPV infection. However, a reliable assay of this kind still does not exist because of the difficulty of obtaining viral antigens by traditional purification methods (5). In our study, similar to others (3,25,29), an extensive number of tests were necessary to find the optimal antigen dilution, which was found to be 10 lg/mL of vaccine diluted in carbonate bicarbonate buffer. The adoption of a temperature of 4C brought on a significant improvement in specificity. In others words, this reduced false positives in the negative samples and increased intra-assay reproducibility. This study, like others (3), also came across another obstacle: the low ranges of optical densities seen mainly in the IgA assay. Studies have demonstrated that specific IgA for HPV VLPs correlates with specific IgG or the DNA-HPV detection (23). However, such studies have concentrated only on the serum IgG responses to HPV-16 and -18 (12,16,24).
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Additionally, it is important to recognize that IgA is one of the most abundant isotypes in serum and plays an important role in the protection against pathological agents mainly at genital mucosal sites (10,11,23). Other studies (15,16,23,24) have similarly observed that IgG and IgA were detected much more frequently in positive samples than in negative samples. Our results also observed that the detection of antibodies against HPV was strongly correlated with the presence of DNA-HPV, confirming the association between serum immunoglobulin responses and DNA-HPV. Our results also conclude that the accuracy of the test was higher for IgG (88/93%), 16/18HPV, respectively than IgA (73%/93%), 16/18HPV, respectively. This may be explained by the fact that IgA remains in the serum for a shorter period of time (16,23). The literature reports that IgG seroconversion against HPV occurs within 6–12 months after infection and that IgG response remains high over a long period of time (12,23). Thus, the duration of IgA in sera and cervical secretions is shorter than the duration of serum IgG (23). Earlier studies have demonstrated that a substantial proportion of women with cervical HPV infection do not seroconvert (3,14,23,31). Carter et al. (3) reported that approximately 60% of their subjects seroconverted within 18 months. These data suggest that development of antibodies at a detectable level after a natural infection can be a slow process, and it does not necessarily occur in every woman. Several factors may explain this event, such as antibodies to HPV circulate at a low titer, low levels of antibodies need to be boosted over time so a detectable seroconversion may take time, and differences in the host genetic background or immune status could also explain why some individuals failed to seroconvert. Carter et al. (3) also reported that individuals who were repeatedly HPV-DNA negative after seroconversion tended not to have persistent IgG antibodies, and this phenomenon was true for antibody responses against HPV-16, HPV-18, and HPV-6 (3). On the other hand, some studies have shown that failure to seroconvert was associated with having had only one HPV-DNA-positive visit for HPV-16. Seroconversion to HPV-18 was also detected less frequently among women who had only one HPV-18 DNA-positive visit than among women with multiple HPV-18 DNA-positive visits (3,6,34). The detection of HPV-16 antibodies is also associated with HPV-16 DNA persistence (3,6,34). De Gruijl et al. (6) found that persistent HPV-16 DNA and HPV-16 serum antibodies were present in all women who developed severe dysplasia in their study. In our study, the seroconversion was detected in patients with persistent HPV infection (HPV injury) and more than 18 months after the detection of HPV-16/18 DNA to avoid samples from women with transient HPV-DNA infection associated with a failure to seroconvert. We have shown herein that the proportion of women who seroconverted (IgG) was more than 80% after 18 months of DNA detection for HPV-16 (sensitivity 82%) and HPV-18 (sensitivity 100%). The better results of our study also could be explained by the significant improvement the sensitivity of antibody assays and evolution of the method during the last 10 years. In order to establish standards for HPV antibody detection, the World Health Organization has coordinated an interna-
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tional collaborative study (8). The results of this study suggest that the establishment of international standard reagents for antibodies to HPV would promote the comparison of HPV antibody measurements between assays and laboratories worldwide. HPV standard reagents for serology will be essential to improve the sensitivity of antibody assays, the consistency of results worldwide, as well as improving interlaboratory and interassay variability, as recommended in comparative studies (29). Besides this, standardization would increase assay performance, allow valid comparison of results between laboratories, and facilitate monitoring of vaccine effectiveness (13,14,23,25). Until now, serology has played no role in the diagnosis of HPV infections, despite serological procedures for the disclosure to and infection with viral pathogens being the usual mode of documentation of viral infection (3,5). There is no classical ‘‘gold standard’’ for measuring HPV serological responses, and different assays may be preferable depending on the questions being asked. The presence of HPV antibodies in the bloodstream of individuals is mostly a marker for past infections, not necessarily correlating to present infection or disease. HPV seropositivity may result from a prior viral infection that has been efficiently cleared, as well from an asymptomatic infection. Consequently, serological assays have not proven their clinical diagnostic value thus far (25). Nevertheless, HPV serology has proven valuable in epidemiological studies based on seropositivity for HPV to understand the natural history of infections and cancer. Importantly, antibody-based immune responses to HPV have contributed notably to understanding the protection provided by neutralizing antibodies, and have allowed for important advancements in the development of a vaccine (13,25). In the context of vaccination, serological assays could be used to screen subjects for suitability and evaluate seroconversion in the vaccine. Moreover, the use of international standardization will guarantee a universal protocol (17,20,25,29,30). In conclusion, for assessment of cumulative exposure to genital HPV infection prior to vaccination, the preferred assay to use may be the VLP ELISA, since it is the most commonly used as well as the easiest to find and least expensive. Thus, ELISA may be useful as a complementary diagnostic tool to the well-established PCR method. Besides this, a standardization of ELISA could be used to monitor the performance of HPV vaccines around the world. However, the availability of high-quality VLPs may be a limiting factor. Some form of quality control for both the vaccine and VLPs needs to be established such as monitoring vaccine performance in laboratories: this would be particularly useful in the attempt to study immune correlates of protection. Acknowledgments
Financially supported by the FAPESP (Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo). Process number: 2011/19960-9. Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to: Dr. Ana Katherine Gonc¸alves Department of Obstetrics and Gynecology Universidade Federal do Rio Grande do Norte Rua General Cordeiro de Farias S/N Petropo´lis Natal–RN 59012-570 Brazil E-mail: [email protected]
