Intralesional Injection of Mycobacterium w Vaccine vs Imiquimod, 5%, Cream in Patients With Anogenital Warts: A Randomized Clinical Trial

Importance  Intralesional antigen therapy has been used in the treatment of anogenital warts (AGWs), but it has not been compared with existing therapies. Evidence of its efficacy is not strong.

Objective  To compare the efficacy and safety of intralesional Mycobacterium w (Mw) vaccine with that of imiquimod, 5%, cream in the treatment of AGWs, as well as changes in human papillomavirus (HPV)-6 and HPV-11 viral loads.

Design, Setting, and Participants  A double-blind randomized clinical trial was conducted in New Delhi, India, between February 2009 and July 2012 and included a 3-month follow-up. Of 159 patients with AGWs who were screened, 89 were randomized.

Interventions  Patients received either imiquimod, 5%, cream and an intralesional vehicle (imiquimod group: 44 patients) or vehicle cream and intralesional Mw vaccine (Mw group: 45 patients).

Main Outcomes and Measures  The primary end point was complete clinical remission of visible AGWs. Secondary measures included the percentage of reduction in the surface area of AGWs and viral load for HPV-6 and HPV-11. Viral load was measured by real-time quantitative polymerase chain reaction.

Results  In the intention-to-treat analysis, 59% (n = 26) of the patients in the imiquimod group and 67% (n = 30) of those in the Mw group had complete resolution (P = .52). Eighteen HPV genotypes, including high-risk genotypes, were detected, with no significant differences between the treatment groups (all P > .05). There was a significant decline in the mean viral loads of HPV-6 (from 0.011 × 10⁸ to 0.00000154 × 10⁸ copies/mg of tissue; P = .003) and HPV-11 (from 0.121 × 10⁸ to 0.017 × 10⁸ copies/mg of tissue; P = .03) after treatment in the Mw group but only in the viral load of HPV-6 (from 1.41 × 10⁸ to 0.004 × 10⁸ copies/mg of tissue; P = .01) in the imiquimod group. There was no recurrence of AGWs in patients with complete clearance at the 3-month follow-up and no serious adverse events.

Conclusions and Relevance  Imiquimod, 5%, and the Mw vaccine were equally effective in achieving clinical and virologic clearance for HPV-6. A significant decline in the HPV-11 viral load was achieved only with the Mw vaccine. Efficacy and safety of intralesional Mw vaccine is comparable to that of imiquimod, 5%, in treatment of AGWs.

Trial Registration  ctri.nic.in Identifier: CTRI/2009/091/000055

More than 120 papillomaviruses are known to infect humans. Of these, more than 40 can infect the anogenital tract leading to external anogenital warts (AGWs) and anogenital cancers.¹ Human papillomavirus (HPV) is one of the leading causes of sexually transmitted infections in the world.² Because of suboptimal efficacy, associated adverse effects, and recurrences associated with available treatments, the management of AGWs remains unsatisfactory.

Activation of cell-mediated immunity appears to be the primary mechanism responsible for the regression and clearance of HPV-related lesions, including AGWs. Therefore, manipulating the immune system, especially cell-mediated immunity, to achieve a therapeutic response against AGWs seems logical. Topical imiquimod stimulates both innate and adaptive immune responses via toll-like receptors, inducing macrophages to secrete cytokines such as interleukin 2 (IL-2), interferon alfa-1 and -2, interferon beta, interferon gamma, and transforming growth factor α. Imiquimod, 5%, is accepted as the first-line treatment for AGWs. Intralesional injections of skin test antigens and vaccines have been reported³ to be effective in the treatment of cutaneous warts and AGWs. Double-blind randomized trials that explore the efficacy and safety of these modalities of treatment are not available.

Mycobacterium w (Mw; revised nomenclature, Mycobacterium indicus pranii) is a nonpathogenic, soil-derived, rapidly growing atypical Mycobacterium. Polyphasic taxonomic studies⁴ have shown that it is a distinct species within the Mycobacterium avium complex. A heat-killed Mw vaccine containing 0.5 × 10⁹ bacilli per 0.1 mL is used as an adjunct to multidrug therapy in patients with multibacillary leprosy. The vaccine generates strong cytokine responses involving IL-2, IL-4, IL-5, and interferon gamma.⁵ In addition to leprosy, Mw vaccine has been studied^6-11 for treatment of tuberculosis and certain cancers.

The Mw vaccine has an immunomodulatory action and cytokine response similar to that of imiquimod. In a recent cohort study, Meena et al¹² reported the complete clearance of cutaneous warts in 83% of 40 patients who received intralesional Mw vaccine. A previous pilot study¹³ confirmed the safety and efficacy of intralesional Mw vaccine therapy for AGWs. We report the results of a randomized trial comparing topical imiquimod, 5%, cream with intralesional Mw in the treatment of AGWs.

Patients were recruited by the investigators (V.K.S, K.K.V., and S.G.) for the trial from the Department of Dermatology and Venerology, All India Institute of Medical Sciences, New Delhi. Written informed patient and parental (for patients aged 12-18 years) consent was obtained. Participants did not receive financial compensation. The study was approved by the institutional ethics committee of the All India Institute of Medical Sciences, and a data and safety monitoring board of 4 experts was constituted.

The inclusion criteria were presence of 1 or more AGW, surface area of 10 mm² or greater of the AGWs, otherwise apparently healthy, age 12 years or older, and untreated for the past 4 weeks. The exclusion criteria were pregnancy and lactation, human immunodeficiency virus seropositivity, a history of cervical intraepithelial neoplasia or cervical carcinoma, any significant systemic illness, substance or alcohol dependence, and an inability to visit the clinic for the duration of the trial.

Sample size calculation was done on the basis of our hypothesis that intralesional Mw vaccine would achieve 50% greater clearance compared with imiquimod, 5%, cream. With expected clearance rates of 40% and 60% in the imiquimod, 5%, cream and Mw vaccine treatment groups, respectively, and at a 5% level of significance with 80% power, a sample size of 48 was calculated for each group.

All of the lesions were photographed at baseline and then at monthly intervals until completion of the follow-up period. Patients were examined at baseline, once every 2 weeks for up to 20 weeks (treatment phase), and then monthly for 3 months during the follow-up phase. The number and surface area of the warts were recorded. Patients were randomized to receive either imiquimod, 5%, cream and intralesional vehicle injections (imiquimod group) or vehicle cream and intralesional Mw vaccine injections (Mw group).

Block randomization with a block size of 6 was performed by a person not involved in the trial. Investigators received prenumbered opaque envelopes containing the study material. The vehicle and drug preparations were identical in appearance. Investigators (clinical and laboratory), patients, and the biostatistician were blinded for the trial intervention. Patients applied imiquimod, 5%, or vehicle cream overnight, 3 times a week for 16 weeks, regardless of the clearance of warts. In addition, participants received 0.1-mL intradermal injections of the Mw vaccine and vehicle on both shoulders at baseline to sensitize them to the vaccine and improve the local immune response to intralesional therapy. Subsequently, intralesional injections were administered every 2 weeks until complete clearance of AGWs (primary end point) or 16 weeks (8 injections), whichever occurred first. If there were significant local or systemic reactions persisting until the next scheduled injection, both the topical treatment and intralesional injection were withheld for a maximum of 2 weeks. Time beyond this period was considered a protocol violation, and the patient was excluded from further trial intervention. The primary end point was clinical resolution of AGWs, and the secondary end point was a clearance of AGWs sustained for at least 3 months following complete resolution.

At baseline, a 5-mm punch biopsy sample was collected from the AGWs and divided into 2 samples: 1 for HPV typing and viral load and the other for histopathologic confirmation. After completion of the treatment phase, a 3-mm biopsy sample was obtained for HPV typing and viral load from the residual AGW or, in case of complete clinical clearance, from the skin adjacent to the previous biopsy site. A biopsy specimen was collected in sterile phosphate buffer saline for HPV DNA testing and stored at −70°C until use. The DNA extraction method of Gravitt et al¹⁴ was used, and DNA was stored at −20°C until amplification for HPV detection and typing.

Detection and typing of HPV were conducted using a genotyping test (Linear Array; Roche Molecular Systems Inc). The test detects 37 HPV genotypes, including 22 high-risk and 15 low-risk types.^6,15

The viral load of HPV-6 and HPV-11 was determined by real-time quantitative polymerase chain reaction (ABI Prism 7500 Sequence Detection System; Applied Biosystems Inc). TaqMan real-time polymerase chain reaction assays were developed for detecting viral loads in the AGW samples. The reaction was based on the E6 gene (HG793938; GenBank) for HPV-6 and the E7 gene (KC329878; GenBank) for HPV-11.¹⁶ A human β-globin gene target was coamplified with that for HPV to determine the adequacy of the specimen and the extraction process and rule out any inhibition of amplification.¹⁷ The constructed cloned plasmid, containing the target gene at a logarithmic dilution range from 10⁷ to 10 copies, was included in duplicate with each run to generate a standard curve.

The data were analyzed using Stata, version 11 (Stata Corp), and are presented as mean (SD), median, range, and frequency. Intention-to-treat analysis was done by carrying forward the last observation of the participants. Categorical variables were compared in 2 groups using the χ² test and Fisher exact test. Continuous variables with a normal distribution were compared between the 2 groups by an independent 2-tailed, unpaired t test, and nonnormal variables were evaluated using the Wilcoxon rank sum test. The effect of treatment within groups was analyzed by the Wilcoxon signed rank test. Differences were considered statistically significant at P ≤ .05.

The study was carried out between February 12, 2009, and August 5, 2012. A total of 159 patients with external AGWs were screened, and 70 of these individuals were excluded for various reasons (Figure 1). The trial was stopped after completion of the period sanctioned by the sponsors. The 89 patients (71 male and 18 female) who met the inclusion criteria were randomized. Forty-four (34 male and 10 female) patients were randomized to receive imiquimod, 5%, cream and vehicle injection (imiquimod group), and 45 (37 male and 8 female) individuals received vehicle cream and Mw vaccine injections (Mw group). Three (7%) patients in the imiquimod group and 6 (13%) patients in the Mw group withdrew, were lost to follow-up, or defaulted before resolution of the AGWs during the treatment phase. Differences between the baseline characteristics of the groups were not significant (Table 1).

Of the 44 patients in the imiquimod group, 26 patients (59%) showed complete clearance and 9 (20%) patients each had 75% or more to less than 100% resolution and less than 75% resolution to no response or worsening, respectively. Of the 45 patients in the Mw group, 30 patients (67%) showed 100% resolution, 7 patients (16%) had 75% or more but less than 100% resolution, and 8 patients (18%) had less than 75% resolution to no response or worsening (Table 2). The mean resolution of AGWs in the imiquimod group was 85%; resolution in the Mw group was 83% (Figure 2). The initial clearance was faster in the imiquimod group compared with the Mw group, although there was no significant difference in the final outcome (Figure 3) and adverse effect profile (severity) (Table 2). The common adverse events in both of the groups were erosions and ulceration, constitutional symptoms, pruritus, and pain. Nodule formation as well as edema and swelling were significantly more common in the Mw group, whereas erythema was more commonly seen in the imiquimod group. Genital discharge and vitiligolike depigmentation were seen only in the imiquimod group.

Human papillomavirus DNA was detected in 84 of 89 patients (94%); HPV-6 and HPV-11 were the most common types (Table 1). High-risk HPV types were also detected. Infections with more than 1 HPV type were detected in 28 patients (31%) (Table 1).

Thirty-seven patients did not consent for postintervention biopsy (imiquimod group, 16; Mw group, 21), so posttreatment HPV genotyping and viral load could be performed only in 52 patients. We compared the preintervention and postintervention viral loads of 26 patients with HPV-6 (imiquimod group, 15; Mw group, 11) and 24 patients with HPV-11, for whom both preintervention and postintervention biopsy samples were available. After completion of the treatment phase, of 52 postintervention biopsy samples, 30 (imiquimod group, 15; Mw group, 15) were HPV negative by polymerase chain reaction, and all of them showed 100% clinical clearance. On comparing pretreatment and posttreatment viral loads, there was a statistically significant decline in the postintervention viral load of HPV-11 in the Mw group but not in the imiquimod group. For HPV-6, the decline was significant in both of the groups (Table 2).

The response to both treatment modalities was good, with no significant difference in the final outcome. Imiquimod, 5%, cream provided faster initial clearance, which may be the result of the difference in the treatment protocol between the agents. Patients received the first intralesional injections of Mw at week 2 and then once every 2 weeks; topical application of imiquimod, 5%, cream was started from day 1 at a higher frequency of 3 times a week. There was no recurrence in patients showing complete clearance at the 3-month follow-up; however, this finding is likely to be an overestimation of sustained clearance rate because AGWs are known¹⁸ to recur several months after complete remission.

Serious adverse events, such as life-threatening reactions leading to hospitalization, disability, or death, were not seen. The adverse events were either in the form of local immunologic or irritant reactions or systemic and constitutional symptoms, such as fever and body ache.

Human papillomavirus has evolved to develop several mechanisms to evade host immune responses, which are crucial for its persistence in the human host. The current emphasis of new drug development for treatment of HPV relies on immunomodulation rather than physical or chemical destruction of visible exophytic growths. The basis of the use of an unrelated antigen or vaccine for HPV-induced lesions was derived from experimental studies by Frazer et al.¹⁹ They reported that the E7 tumor antigen of HPV-16, expressed in keratinocytes of mice transgenic for E7 tumors, was only weakly immunogenic when transplanted into a naive host mouse. However, when a strong proinflammatory signal in the form of a Listeria monocytogenes injection was delivered simultaneously, there was a specific rejection of E7 transgenic skin of the host immune system. A second similar graft was also promptly rejected without coadministration of L monocytogenes. This finding suggests that delivering a strong proinflammatory signal helps in eliminating HPV infection. The Mw vaccine has been reported⁶ to induce a type 1 helper T-cell response and activate macrophages in an animal model. Our hypothesis is that when the Mw vaccine is injected into HPV-infected tissue, it generates strong proinflammatory signals and attracts antigen-presenting cells, which process both the injected antigen (which acts as an adjuvant) and the HPV antigen present in the infected tissue. The cells then migrate to regional lymph nodes and present these antigens to CD4⁺ and CD8⁺ lymphocytes, which further expand and migrate to the infection site and induce CD4⁺ lymphocyte–mediated CD8⁺ cytotoxic damage of HPV-infected cells.

In the present study, the clearance rate with imiquimod, 5%, was relatively higher than that previously reported. Additionally, most patients in our study were male, and response rates with imiquimod, 5%, are generally better in females. Beutner et al²⁰ reported clearance rates of 42% in males compared with 64% in females. In the present study, 77% and 82% of the patients in the imiquimod and Mw groups, respectively, were male.²¹ The higher clearance rates with imiquimod in our study may have been the result of needle-prick trauma during concomitant injection of the vehicle, which may have added to the inflammation and enhanced the immune response induced by imiquimod, 5%.

There are several reports on the use of intralesional antigen and vaccine therapies in cutaneous warts, but none of them was a double-blind randomized trial, and only 3 studies^3,13,22 looked at the effect of these therapies on AGWs. A retrospective analysis²² using immunotherapy with mumps as well as Candida and/or Trichophyton skin test antigens in patients with AGWs reported complete resolution in 5 of 10 patients who completed the therapy. Candida and/or Trichophyton antigens may be useful in countries with a low prevalence of tuberculosis because Mw may interfere with the purified protein derivative screening test. In a pilot study¹³ of intralesional injection of Mw, 8 of 10 patients with AGWs had complete resolution. Eassa et al³ reported complete clearance in nearly half of the pregnant women with AGWs who received intralesional purified protein derivative of Mycobacterium tuberculosis. The clearance was significantly associated with strong tuberculin test positivity.

To our knowledge, this is the first report in which HPV genotyping was done in Indian patients with AGWs. As expected, the predominant types were HPV-6 and HPV-11, which separately or combined occurred in 82% of the patients. Twenty-eight (31%) samples showed infection with multiple HPV types. There was no statistically significant difference in the response rates in infection with particular or multiple HPV types.

The decline in HPV-6 viral load was more impressive than for HPV-11. The Mw group showed a significant decline in both HPV-6 and HPV-11, but the imiquimod group experienced a significant decline only for HPV-6. A decrease in the posttreatment viral load correlated with clinical resolution.

Although it is invasive and associated with local immunologic reactions, intralesional Mw vaccine therapy is as effective as imiquimod, 5%, in the treatment of AGWs and results in elimination of HPV in the lesion. We will endeavor to further study the efficacy of the Mw vaccine in AGWs that fail to respond to imiquimod, 5%, and other conventional therapies.

Accepted for Publication: April 2, 2014.

Corresponding Author: Somesh Gupta, MD, Department of Dermatology and Venereology, Room 4066, Fourth Floor, Teaching Block, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029, India (someshgupta@hotmail.com).

Published Online: August 6, 2014. doi:10.1001/jamadermatol.2014.794.

Author Contributions: Drs Dar and Gupta had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Sharma, Verma, Gupta.

Acquisition, analysis, or interpretation of data: Kumar, Dar, Saldiwal, Varma, datt Upadhyay, Talwar, Verma, Dwivedi, Raj, Gupta.

Drafting of the manuscript: Kumar, Dar, Saldiwal, Varma, datt Upadhyay, Talwar, Raj, Gupta.

Critical revision of the manuscript for important intellectual content: Sharma, Verma, Dwivedi, Gupta.

Statistical analysis: datt Upadhyay.

Obtained funding: Sharma, Gupta.

Administrative, technical, or material support: Kumar, Dar, Saldiwal, Varma, Talwar, Verma, Dwivedi, Raj, Gupta.

Study supervision: Dar, Sharma, Verma, Gupta.

Conflict of Interest Disclosures: None reported.

Funding/Support: This study was supported in part by the Department of Biotechnology, Government of India.

Role of the Sponsor: The sponsor had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Additional Contributions: Shinjini Bhatnagar, PhD, and Dharmendra Kumar, MCA, from the All India Institute of Medical Sciences, assisted with randomization; V. G. Ramachandran, PhD, University College of Medical Sciences, and Bindu Dey, PhD, Government of India, provided technical guidance; Vishal Madan, MRCP, Consultant Dermatologist, edited the manuscript; and Arbind Kumar, MSc, All India Institute of Medical Sciences, assisted with the study. No financial compensation was given for these services.