Somesh Gupta, MD, DNB, MNAMS, Professor, Department of Dermatology & Venereology, AIIMS, New Delhi

1. Immunotherapy for Genital
HPV
Somesh Gupta, MD, DNB, MNAMS
Professor,
Department of Dermatology & Venereology
AIIMS, New Delhi
Regional Director, IUSTI-AP

2. CONFLICTS OF INTERESTS- NONE
Funding SourceDepartment of Biotechnology, Govt. of India

3. UAA/AAUS GUIDELINEFOR STIS
International Journal of Urology

4. Level of evidence (LoE)
Type of evidence LoE
Evidence obtained from meta-analysis of randomised trials 1a
Evidence obtained from at least one randomised trial 1b
Evidence obtained from at least one well-designed controlled study without
randomisation
2a
Evidence obtained from at least one other type of well-designed quasi-experimental
study.
2b
Evidence obtained from well-designed non-experimental studies, such as
comparative studies, correlation studies and case reports.
3

5.  HPV can infect Skin, Uterine Cervix, the lower genital
region, the anus, oropharynx
 Transmission
 Skin-to-skin contact through sexual intercourse
 Oral sex
 Anal sex
 Other contact involving the genital area.
 The virus may remain latent until a trigger causes
replication of viral DNA, leading to anogenital wart.
 More than 40 HPV types - genitourinary tract and anal
region
 High Risk/ Low Risk

6. 55%
42%
26%
11%
0%
15%
30%
45%
60%
HPV 6 HPV 11 High Risk Other Low Risk
HPV Types in AGW (n=89)

7. HRQOL in patients with Viral
STIs
0.
25.
50.
75.
100.
125.
Physical Domain Psychological Social Relationship Environmental
QOL
Controls
HPV
HSV
HIV
Mixed
Raj R, Sreenivas V, Mehta M, Gupta S. Health-related quality of life in Indian patients with three viral sexually
transmitted infections: herpes simplex virus-2, genital human papilloma virus and HIV.Sex Transm Infect. 2011
Apr;87(3):216-20.

8. Predisposing factors
 Early age of onset of sexual activities
 Multiple sexual partners
 A history of STDs
 An early age of pregnancy
 Tobacco use
Albero G, Castellsaguams H. Human papillomavirus: cliniircumcision and
genital human papillomavirus: a systematic review and meta-analysis. Sex
Transm Dis. 2012 ;39:104-13.
Circumcision: partially protective

9. Anogenital warts
 Natural course-
 resolves on its own
 remains unchanged
 increases in size or number
 Spontaneous resolution within 2 years - in 65-78% of
warts

10. Risk factors for persistent HPV
infection
 Multiple sex partners
 Sex at an early age
 History of sexually transmitted infections
 Smoking

11. Clinical Types
 Condylomatous, keratotic, papular, and flat.
 Exhaustive proctological examination in
patients suffering from condylomatosis of the
genital area
 Out of 362 patients with both endoanal and
perianal condyloma, only half of them were
detectable by perianal clinical examination,
rest were detected only with the help of
proctoscopic examination

14. Host defence mechanisms
 Regressing Genital Warts- a large infiltrate
comprising of T cells (CD4+ and CD8+) and
macrophages
 Infiltrating lymphocytes express activation
markers, pro-inflammatory cytokines such as
IL-12, TNF-α, and IFN-γ
 Upregulation of the adhesion molecules on the
endothelium of the wart capillaries

15. Expression of E1
& E2- DNA
replication
Expression of E4,
L1 & L2 protein in
differentiated
cells
Assembly and
release
Episomal viral
replication: E6,
E7, (Promote cell
proliferation &
Delay
differentiation)
A6b1 and a6b4
Langerhans Cells
The life cycile of HPV is tied
to the keratinocyte
differentiation program
No blood born phase
Do not cause lyses of
keratinocytes
HPV do not infect and
replicate in Antigen
Presenting Cells (APC)
‘Early’ proteins
localized in nucleus
Delay in the expression of
‘late’ proteins- not
accessible to langerhans
cells

16. Immunotherapeutic
strategies
 Lyses of infected keratinocytes to make HPV ‘Late’
genes available to APCs, mainly located in the
dermis
 Release cytokines that attract Cytotoxic T cells to
the site of HPV infection
 Introduce a strong antigenic substance to attract
APCs in the epidermis- the site of HPV infection
 Deplete T-regulatory cells from the HPV infection
site.

17. Treatment
Physician administered (LE) Provider administered (LE)
Photodynamic therapy (1a) Polyphenon 10% (1a)
Immunotherapy (1b) 5- Fluorouracil (1a)
Cryotherapy (1b) Interferon (1a)
Podophyllin (1b) Imiquimod 5%(1a)
Laser (2) Podophyllotoxin (1b)
Surgery (3) Cidofovir (3)

18. Treatment modality Clearance rate % Recurrence rate%
PDT 85.7 9.38-14.3
Cryotherapy 79-88 25-40
Laser 23-52 60-77
Surgery 72 19-29
Immunotherapy 66.7 No at 3months, Long Term NA
Podophyllin 42 46-60
Imiquimod 56 13-16
Podophyllotoxin 45-77 38-65
Sinecatechecin/ 58 6-9
Polyphenon E 40-50 10
5-FU 10-50 50
Interferon 17-67 9-69

19. Probability of Recurrence in
Anal Warts
10%
14%
16%
20%
30%
33%
15%
20%
28%
0%
10%
20%
30%
40%
One Year Two Year Three Year
Minimal Dise
Moderate Di
Extensive D
Silvera RJ, Smith CK, Swedish KA, Goldstone SE. Anal condyloma treatment and recurrence in HIV-negative men who have sex with men.
Dis Colon Rectum. 2014 Jun;57(6):752-61.

20. Photodynamic therapy
 PDT- photosensitizer-mediated and light-
induced cytotoxicity
 Has been successfully used for the treatment
of AGW
 Lower recurrence rate when CO2 laser
ablation was followed by PDT

21. ALA-PDT
In the epidermis, CD1a+ cells (dendritic ⁄Langerhans cells) slightly
outnumbered those in the dermis.
Giomi B, et al. Immunological activity of photodynamic therapy for
genital warts. Br J Dermatol 2011;164:448-51.

22. 5‐aminolevulinic acid‐based photodynamic therapy for the treatment of condylomata
acuminata in Chinese patients: a meta‐analysis
Photodermatology, Photoimmunology & Photomedicine
Volume 29, Issue 3, pages 149-159, 7 MAY 2013 DOI: 10.1111/phpp.12043
http://onlinelibrary.wiley.com/doi/10.1111/phpp.12043/full#phpp12043-fig-0003

23. Imiquimod LoE1a
 Imiquimod 5% cream
 Imiquimod 3.75% cream, daily application- 43.1% clearance
rate
 Response rates are higher in women than in men (60% vs.
35%)
 Poor response in untreated HIV seropositive patients (≅11%)
(Gibson RJ, et al. AIDS 1999;13:2397-404)
 Low relapse rate for imiquimod (6.3% vs. 25% for ablative
therapies)
 Imiquimod and a combination of imiquimod and ablative therapy
have been found superior to ablative treatment alone (Schfer H,
et al. Eur J Dermatol 2006; 16:642-8.)
 If there is partial response in first 12 weeks therapy, a further
course will produce complete clearance in another 27%

24. Imiquimod
Study
# Patients
Receiving
Imiquimod
5% Cream
Dosing
Interval
Complete
Clearance
Rate
Length of
Treatment
Recurrenc
e Rates
After
Treatment
Beutner,
Spruance
et al.10
108
3
times/week
37% 8 weeks
19% at 10
weeks
Garland et
al.11 943
3
times/week
47.8% 16 weeks
8.8% at 3
months
23% at 6
months
Edwards et
al.12 109 / 311
3
times/week
50% 16 weeks
13% at 3
months
Beutner,
Tyring et 94 / 279 Once daily 52% 16 weeks
19% at 3
monthsSkin Therapy Letter.com

25. Adverse Reactions
 A moderate to severe local reaction is seen in
all patients and clearly associated with efficacy
 Erythema, induration, burning and itching are
common
 Ulceration and erosions can occur in some

28. Laser ablation+low dose
cycloposphamide LoE1b
 Cao et al. : combined CO2 ablation with low
dose cyclophosphamide- compared with CO2
ablation alone and CO2 ablation + high dose
cyclophosphamide
CO2 +Placebo CO2 + Low dose
cyclophosphamide
N 26 52
Recurrence 21 (81%) 9 (17%)
Cao et al. Clinical Immunology 2010;136:21-29.

29.  FOXP3+ regulatory T (Treg) cells are
accumulated in genital warts 
immunosuppression
 Low dose of cyclophosphamide selectively
target Treg cells  enhancing the function of
HPV-specific T cells and NK cells  efficient
clearance of HPV infection
Zhao J, Zeng W, Cao Y, Liang X, Huang B. Immunotherapy of HPV infection-caused genital warts using low dose cyclophosphamide.
Expert Rev Clin Immunol. 2014 Jun;10(6):791-9.

30. Indications for Intralesional
Immunotherapy
 1. Recalcitrant warts
 2. Recurrent warts
 3. Extensive warts

31. Killed Mycobacterium w vaccine
 Mycobacterium w is a nonpathogenic, soil
derived, rapidly growing, atypical
Mycobacterium
 Shares a number of common B and T cell
determinants with M. leprae and M.
tuberculosis
 It generates strong cytokine (IL-2, IL-4, IL-5,
IFN-) and T cell responses
Gupta S, et al. Intralesional immuinotheraphy with killed Mycobacterium w
vaccine for the treatment of anogenital warts: an open label pilot study. JEADV
2008; Vol 22, Issue 9, start page 1089

34. Self-resolving
Granulomatous
dermatitis at the site
of intralesional
injection
Epithelioid cell granulomas with giant cells and eosinophils
Stain for AFB: Negative

36. INTRALESIONAL INJECTION OF MYCOBACTERIUM W
VACCINE VS IMIQUIMOD, 5%, CREAM IN PATIENTS WITH
ANOGENITAL WARTS: A RANDOMIZED CLINICAL TRIAL.
Kumar P, Dar L, Saldiwal S, Varma S, Datt Upadhyay A, Talwar D,
Sharma VK, Verma KK, Dwivedi SN, Raj R, Gupta S. JAMA
Dermatol. 2014 Oct;150(10):1072-8.

37. Objectives
 To compare the efficacy and safety of IL
injection of Mycobacterium w (Mw) Vaccine
and 5% Imiquimod cream in the management
of External AGW.
 To compare the effect of both the treatments
on reduction of viral load of HPV 6 and 11.

38. Materials and Methods
 Inclusion criteria:
 patients with one or more AGW,
 surface area of warts 10mm2 or more,
 otherwise apparently healthy,
 more than 12 years of age,
 had not received any treatment during the past 4 weeks.
 The exclusion criteria
 pregnant and lactating women,
 HIV seropositive patients,
 Known cases of cervical intraepithelial neoplasia or cancer cervix,
 having significant systemic illness,
 unable to visit two-weekly for trial intervention.

39. Prior Sensitization with 0.1 ml of Mw
or vehicle vaccine on both deltoids
Week 0
Intralesional injection (up to 0.1 ml) of
vaccine or vehicle into the lesions
Week 2
Repeated at 2 weekly interval – 8
injections
Thrice weekly
imiquimod or vehicle
cream was started
from Week 0

40. HPV typing and Viral Load
 Sample collection: Biopsy Samples of anogenital
warts were collected in sterile PBS and transported to
the laboratory and stored at -700C until use. Five mgs of
each specimens were weighed and placed into 5 μl of
TE (Tris- EDTA Buffer) Solution.
 DNA Extraction Methology: The method of Gravitt et
al. (J. Clin Microbiol, 36:3020-3027, 1998) was followed
 HPV detection and typing: by using Linear Array HPV
genotyping Test Kit (Roche Molecular System Inc.,
Alameda, California, (USA)

41. Results

42.  HPV DNA was detected in 84 (94.38%) of 89
patients by PCR reverse line blot assay
 High risk HPV types were found in 23 (25.8%)
patients
 Infection with more than one HPV type was
detected in 22(24.7%)

43. Imiquimod Mw P value
Number of patients 44 45
Males (%) 34 (77.3%) 37 (82.2%) 0.561
Age in Years Mean±S.D. 29.45±10.09 26±8.38 0.53
Lifetime number of sex partners, Mean±S.D. 2.1±1.1 2.2±1.3 0.85
Number of sex partners in last three month, Mean±S.D. 0.8±0.71 0.84±0.8 0.95
Number of sex partners in last one year, Mean±S.D. 1.45±1 1.4±1.1 0.59
H/o anogenital warts in regular partner
Yes
No
Don’t Know
7
33
4
6
32
7
0.83
Duration of anogenital warts in months, Median (min-max) 4.5(0.5-24) 4(0.6-84) 0.53
Number of warts, No. of patients (%)
1-5
6-10
11-20
>20
3 (6.8%)
8 (18.2%)
14 (31.8%)
19 (43.2)
6 (13.3%)
7 (15.6%)
9 (20%)
23 (51.1%)
0.482
Baseline Characteristics

44. HPV typing
Imiquimod Mw P value
HPV type 6, Number of patients (%) 25(56.8%) 24 (53.3%) 0.741
HPV type 11, Number of patients (%) 20 (45.5%) 17 (37.8%) 0.463
HPV types High risk, number (%) 11(25%) 12(26.7%)
16*
18*
31*
33*
35*
51*
52*
56*
58*
59*
66*
73*
4(9.09%) 1(2.23%) 0.15
1(2.27%) 3(6.67%) 0.31
1(2.27%) 1(2.23%) 0.98
0(0%) 1(2.23%) 0.32
0(0%) 1(2.23%) 0.32
0(0%) 1(2.23%) 0.32
0(0%) 1(2.23%) 0.32
1(2.27%) 0(0%) 0.30
1(2.27%) 0(0%) 0.30
1(2.27%) 3(6.67%) 0.31
1(2.27%) 0(0%) 0.30
1(2.27%) 2(4.45%) 0.57
Other low risk HPV types, Number (%) 5(11.36%) 5(11.12%)
42
61
3 0 0.07
1 1 0.98

45. Response to Treatment
59%
21% 21%
100%
67%
16% 18%
100%
0%
25%
50%
75%
100%
Complete Clearance >75-<100% <75% Sustained Clearance*
Imiquimod
Mw
*For sustained clearance, denominator is number of patients who had 100% resolution
at the end of treatment and came for at least 3-mnoth post-treatment follow-up

46. Percentage resolution in the area of warts
in Imiquimod and Mw groups
30.22
20.00
58.52
41.34
68.86
53.56
71.93
62.71
75.68
67.22
78.40
73.22
80.90
76.24
85.04
79.51
84.65
83.23
P= 0.063
P= 0.026
P= 0.080
P= 0.221
P= 0.259
P= 0.532
P= 0.733
P= 0.595
P= 0.651

47. Imiquimod Mw vaccine
Number of days taken for
complete resolution,
Mean±S.D.
46.4±59.0 53.5±66.6 P=0.459

49. Adverse Events
6.8
50.
29.6
13.6
93.2
11
60
20
9
89
0.
25.
50.
75.
100.
None Mild Moderate Severe All
Imiquimod
Mw vaccine

51. HPV 6 Viral Load
0.
4000000.
8000000.
12000000.
16000000.
Imiquimod
Mw
15500000
1094605
10722.
223.6
HPV-6-Pre-treatment Viral Load
HPV-6 post-treatment viral load
P=0.0010
P=0.0058
Copies/mg

52. HPV-11 Viral Load
0
15000000
30000000
45000000
60000000
Imiquimod Mw
50500000
12100000
41500000
1747291
HPV-11-Pre-treatment Viral Load
HPV-11 post-treatment viral load
P=0.5136
P=0.0329
Copies/mg

53. After 8 weeks, 4 doses
Courtesy: Dr. Saurabh Singh, PGI, Chandigarh, India

54. Extra-Genital Warts
Baseline After 3 injAfter 1 inj
Singh S, Chouhan K, Gupta S. Intralesional immunotherapy with killed Mycobacterium indicus pranii vaccine for the treatment of extensive cutaneous
warts. Indian J Dermatol Venereol Leprol. 2014 Nov-Dec;80(6):509-14.

55. At 5 months
Singh S, Chouhan K, Gupta S. Intralesional immunotherapy with killed Mycobacterium indicus pranii vaccine for the treatment of extensive cutaneous
warts. Indian J Dermatol Venereol Leprol. 2014 Nov-Dec;80(6):509-14.

56. Singh S, Chouhan K, Gupta S. Intralesional immunotherapy with killed Mycobacterium indicus pranii vaccine for the treatment of extensive cutaneous
warts. Indian J Dermatol Venereol Leprol. 2014 Nov-Dec;80(6):509-14.

57. Why injection of unrelated antigens induce
immune response against HPV?
Non E7 transgenic
Not rejected
Rejected Rejected
E7 transgenic
Skin graft
2nd Skin
graft
Listeria or
endotoxin
Tindle RW. Immune evasion in human papillomavirus-associated cervical cancer.
Nature Reviews/Cancer. 2002, Vol. 2, Page 59.

58. HPV
wart
Danger
Signals
Immature
Dendritic
Cells
Mature
Dendritic
Cells
Lymph Node
Help
KillAPC
Danger
Signals
Epitope
TCR
MHC II
MHC I
CD28
B 7
CD 40
CD 40

59. Conclusions…….
 The clearance rates of available therapeutic agents – 40-
60%
 Recurrence rates- 6%-80%
 Immunomodulatory therapies appear to be associated with
less frequent recurrences than ablative therapies (LoE 2,3)
 Recurrences can be treated with same regimen used
earlier
 Those who fail with one therapeutic agent/procedure, may
respond to alternative treatment
 First line of treatment should be less expensive, least
invasive and least toxic

60. Conclusions
 Enough studies to suggest that Intralesional
immunotherapy for cutaneous and genital warts is
effective and safe
 All antigens and vaccines appear to give similar results
 There is no need for pre-sensitization by giving shoulder
injections
 Most responders respond within 3-4 injections
 Tentative upper limit for injections is 8
 The frequency of injection once every two weeks,
though other protocols need to be studied and
compared.

61. Acknowledgement
Pankaj Kumar, MSc;
Dr. Lalit Dar, MD;
Dr. Vinod Kumar Sharma, MD;
Dr. Kaushal Kumar Verma, MD;
Dr. Sada Nand Dwivedi, PhD;
Dr. Saurabh Singh, MD,
Dr. Kavish Chouhan, MD,
DSMB-
Dr. V. Ramesh,
Dr. M. Ramam,
Dr. V. Sreenivas
Randomization
Dr. Shinjini Bhatnagar