The Impact of Targeted Therapy on Microorganism in Patients With Psoriasis
Introduction As numerous studies have identified TNF-α, TH-17 and IL-17 as particularly
major roles governing the inflammatory cascade in psoriasis, target therapies, including
TNF-α and TH-17 antagonists, have represented major breakthrough in the treatment of
psoriasis. It is well known that TNF-α plays an important role in the immune response to
viral infections, which is attributed to direct inhibition of viral replication, killing of
virally infected cells, induction of FasL-dependent CD8+ T-lymphocyte effector pathways
through TNF/TNF receptor 2 interaction and promote inflammatory response
indirectly.Additionally, it has been shown that treatment with TNF-α antagonists is
associated increased viral replication in virus-inoculated rats, progressive loss of CD4+
and CD8+ T cells and decreased level of TNF-α and interferon. A transient decreased T cell
responsiveness was also observed in some patients treated with TNF-α antagonists.10 TNF-α
can inhibit CMV production in certain cell types and the CMV major immediate early promoter
is inhibited by TNF-α. Increased TNF production has been reported during CMV infection,
which also displays anti-CMV activity in vitro.
TNF-α is also involved in direct control of HPV infection by induction of apoptosis of
HPV-infected cells, arresting growth of HPV-infected keratinocytes and down-regulation of
HPV-gene transcription, stimulation of inflammatory response and up-regulation of HLA class
I components in antigen-presenting cells. E6 protein of HPV-16 is known to directly bind to
TNF receptor 1 and abrogate TNF-induced apoptosis of the host cells. Recent studies have
shown a high prevalence of HPV DNA in psoriatic skin and increased HPV5 antibodies in
patients with psoriasis. It seems that psoriatic skin may be specifically permissive for
HPV. Increased pap smear abnormalities has been observed in patients with inflammatory bowel
diseases receiving TNF-α antagonists. However, the specific effects of anti-TNF therapy on
HPV carriage and HPV-associated disease remain unknown.
Epstein-Barr virus (EBV) is associated with several B-cell and epithelial-cell malignancies,
especially in patients under the immunosuppressive treatment. Increased risk of
lymphoproliferative disorders in patients treated with TNF-α antagonists has been addressed.
Case reports of EBV-associated lymphoproliferative diseases that regressed after withdrawal
of anti-TNF-α drugs had been also described. Thus, the concern of the increased risk at
least in part caused by immunosuppression of anti-TNF therapy, which may cause a consecutive
EBV reactivation, emerged but was not proven yet.
Anti-TNF-α drugs, including monoclonal antibody against TNF-α (infliximab and adalimumab)
and soluble TNF receptor (etanercept), are recognized as a risk factor for opportunistic
infections. A recent study demonstrated a higher risk of opportunistic infections for
patients receiving adalimumab than etanercept, with a 10- to 17-fold differences in risk.
However, most knowledge of latent viral reactivation during anti-TNF treatment has come from
studies of patients with inflammatory bowel disease and rheumatoid arthritis. Moreover,
these studies mainly focus on herpesviruses and viral hepatitis. Several case reports in
which EBV, CMV,and HPV infection complicated therapy with TNF-α antagonists in patients with
psoriasis have also been reported.
IL-17 is a proinflammatory cytokine that is produced by a variety of cells, including a
newly identified subset of activated CD4+ T cells (Th17 cells), γδT cells, CD8+ cells and
NKT cells. There is evidence to support a protective role for IL-17 in the host defense
against extracellular bacterial infections, including Klebsiella pneumonia,38 Bacteroides
fragilis and Escherichia coli. A recent study also showed IL-17 is essential for human
immunity against common fungus Candida albicans.
Various bacteria and fungi are reported to be associated with provocation and/or
exacerbation of psoriasis. The strongest evidence exists for the induction of guttate
psoriasis by a preceding tonsilar Streptococcus pyogenes infection. Balci et al also found
significant higher rates of Staphylococcus aureus colonization in lesional skin and nares of
the patients with psoriasis than healthy controls. They also suggested a significant
relationship between PASI scores and both S. aureus cultivation and toxin production in
lesional skin of the patients with psoriasis.
However, the studies investigating the impact of TNF-α, TH-17, IL-17 antagonists and other
targeted agents in patients with psoriasis are limited.
Aim： In Taiwan, the seropositive rate of CMV is approximately 50% for school age children
and more than 90% for adults. EBV causes a persistent infection of B lymphocytes in more
than 90% of the adult population worldwide.46 Thus, the importance of investigating the
influence of target therapies on the latent viruses and other microorganisms can't be
overemphasized. The aim of this study is to prospectively investigate the effect of TNF-α,
TH-17 and IL-17 antagonists administration on the activation of viruses, including CMV, EBV
and HPV and the impact of biologics on the prevalence of surface colonization of
microorganism, including HPV, bacteria and fungi, in patients with psoriasis. The microbial
effect of novel targeted agents, like tofacitinib, secukinumab and apremilast, will also be
Methods Patients Characteristics and Study Schedule We include patients with psoriasis who
will receive and who are currently on TNF-α (etanercept and adalimumab) for psoriasis, in a
tertiary referral centre in northern Taiwan (National Taiwan University Hospital (NTUH))
between 2011 and 2015. We also include patients who have signed informed consent to use or
participate in the clinical trials and are currently using the target therapies of
ustekinumab, secukinumab, tofacitinib (CP690,550), and apremilast. These clinical trials are
approved by the Research Ethics Committee of the NTUH (NTUH-REC: ustekinumab, 200806022M;
secukinumab, 201104022MA; tofacitinib, 201012013MA; apremilast, 201010022M). Patients
themselves or their guardians have signed informed consent for the study protocols of these
new drugs before entrance to this study. All the participants of our study or their
guardians also need to sign another informed consent of this study, which is approved by the
Research Ethics Committee of the NTUH (NTUH-REC No: 201106079RC).
This project consists of two related studies:
1. Patients who are planned to receive targeted agents:
This prospective, observational study includes psoriasis patients who are going to
receive targeted agents. After enrollment, CMV and EBV IgG are checked. Patients
undergo the sampling of blood, eyebrow hairs and skin scales before the initiation of
biologics therapy, 12 and 24 weeks after initiation of the target therapies, 12 weeks
after discontinuation of target therapies. Blood samples are sent to check viral load,
and CMV and EBV viral load are determined by real-time PCR. Bacterial, fungal cultures
and HPV DNA detection of skin scales are also performed. HPV typing is determined by
genechip and semi-quantitative measurement of HPV viral load is done. Demographic data
and the activity parameters of the psoriasis, such as psoriasis area and severity index
(PASI), are collected at baseline and each study visit. The aim of this study is to
investigate the dynamic effect of target therapies administration on the
microorganisms, including CMV, EBV, HPV, bacteria and fungi in patients with psoriasis.
2. Patients who have received targeted therapy The second part of our project, a
case-control study, includes psoriasis patients who have received targeted agents for
more than 3 months. We also recruit age-and disease severity-matched psoriasis patients
who are not treating with targeted agents or other systemic antipsoriatic agents as a
control group. Likewise, after enrollment, CMV and EBV IgG are checked. HPV detection,
bacterial, fungal cultures of the skin scales were done. Comparison of the prevalence
of latent virus, virus reactivation, bacteria and fungi skin colonization between
psoriasis patients who are treating with and without target therapies is performed.
Additionally, the microorganism examinations are repeated again 12 weeks after the
discontinuation of target therapies. The dynamic changes of viral serology, bacterial and
fungal cultivation data after stopping target therapies are analyzed.
The aim of this part of our study was to assess any difference of the status of latent virus
or microorganism colonization in skins between psoriasis patients treated with and without
Scale sampling on the nonlesional skins for HPV The sampling areas will be first cleaned
with Hibitane and gentlely taped with adhesive tapes to remove surface contamination. Then a
superficial skin scraping is taken from the head and neck areas, as far as possible from any
psoriatic plaques, using a fine sandpaper to abrade the stratum corneum and collecting the
debris for HPV detection.
Cultivation of bacteria and fungi on the skin folds After clearing sampling areas with
Hibitane, skin swab samples are taken from skin folds, including retroauricular areas,
inguinal or axillary areas, by using a sterile cotton swab dipped in double-distilled water
and then a gentle swabbing of the skin.
Hair sampling for HPV Eyebrows are plucked with new tweezers used for each subject. Only
hairs containing intact hair follicles are collected for HPV studies.
Determination of viral loads
Quantitative PCR for CMV and EBV viral load Whole blood specimens were stored at − 80 ◦C
before testing. DNA was extracted from 400μL of sample with the MagNA Pure Compact Nucleic
Acid Isolation kit I using the MagNA Pure Compact Instrument (Roche Diagnostics). The volume
of solution was 100μL. Viral DNAs were then amplified using the "in-house" real-time PCR
assays implemented in the laboratory for virological diagnosis. For CMV, real-time PCR
assays based on hydrolysis probe technology were used, as described previously.48 For EBV,
the "in-house"real-time PCR assay using hybridization probes was carried out, as reported
previously.49 CMV amplification was performed on the ABI PRISM® 7500 (Applied Biosystems,
Courtaboeuf, France) using the following cycle parameters: 2 min at 50 ◦C and 10 min at 95
◦C, followed by 45 cycles each consisting of 15 s at 95 ◦C and 1 min at 60 ◦C (ramprates of
0.8 ◦C/s up and 1.6 ◦C/s down; 9600 emulation mode). EBV assay was carried out on the
LightCycler® 1.5 (LC1.5; Roche Diagnostics). EBV assay was performed with the following
cycle parameters: 1 min at 50 ◦C and 8 min at 95 ◦C, followed by 45 cycles each consisting
of 10 s at 95 ◦C, 15 s at 58 ◦C and 15 s at 72 ◦C, and a cooling step of 1 min at 40 ◦C
(ramp rate of 20 ◦C/s). Real-time PCR assays were performed in parallel on the LC480, after
slight adaptations from the initial protocols. Ten microliters of extracted DNA were
amplified in 25μL of mixture containing 1 × LightCycler® 480 Probes Master, 200 nM forward
primer, 200 nM reverse primer, 100 nM hydrolysis probe, 0.38 units uracil-N-glycosylase
(Invitrogen, Cergy-Pontoise, France). For EBV, the hybridization probe labeled with LC-Red
640 was used as a FAM-labeled hydrolysis probe.49 The sequences of all primers and probes
used in LC480 protocols are indicated in Table 1. Cycling conditions on LC480 were as
follows: 2 min at 50 ◦C and 10 min at 95 ◦C, followed by 45 cycles each consisting of 15 s
at 95 ◦C and 40 s at 60 ◦C, and a cooling step of 30 s at 40 ◦C (ramp rate of 4.4 ◦C/s). For
CMV and EBV assays, quantitation was achieved with a standard curve generated from 10-fold
serial dilutions of viral DNA extracted from infected cell cultures and quantified using a
plasmid. In order to detect potential PCR inhibitors, all DNA extracts were tested undiluted
and at a dilution of 1:10. Results were expressed logarithmically (viral copy number/mL).
Given the extraction dilution, the sensitivity threshold was 1.4 logs for all assays.
HPV genotyping and semiquantitative HPV viral load The HPV genechip (Easychip HPV Genotyping
Array, King Car, Taipei, Taiwan) will offer an revert-blot hybridization to detect 39
subtypes of HPV DNA (6, 11, 16, 18, 26, 31, 32, 33, 35, 37, 39, 42, 43, 44, 45, 51, 52, 53,
54, 55, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 72, 74, 82, CP8061, CP8304, L1AE5, MM4, MM7,
and MM8) in a single reaction, as reported by Huang et al. The nested multiplex PCR products
of L1 region of HPV of each specimen will be hybridized with a genechip. The other set of
nested PCR will be also performed for skin typed HPV which consisted of FAP59/64 and
Fap6319R/6085F and DNA sequencing analysis.will be performed for the product to rule out the
presence of coinfection. The genechip is able to detect multiple infection and the
skin-directed PCR sequencing can only detect one serotype of HPV at one time. Internal
Control for extracting of human-genomic DNA
1. . GAPDH, gap-1/gap-2 (ca. ~341 bp)
2. . Beta-globin, KW42/KW29 (ca. ~510 bp)
3. . Beta-globin, KC03/KC04 (ca. ~110 bp)
4. . Beta-globin, KC03/KC05 (ca. ~150 bp)
Strategy of mucosal HPV detection:
Primers mDeAV1 5'-CGTCCM*ARRGGAWACTGATC-3' mDeAV2 5'-GCMCAGGGWCATAAYAATGG-3'-Biotin GAP1:
5'-CCAACTTTCCCGCCTCTCAGC-3' GAP2: 5'-AAAACCGCTAGTAGCCGGGCC-3' KW29:
5'-GGTTGGCCAATCTACTCCCAGG-3' KW42: 5'-GCTCACTCAGTGTGGCAAAG-3'
For the detection of skin typed HPV, the other sets of nested PCR primer will be used as:
FAP6085F：CCWGATCCHAATMRRTTGC FAP6319R：ACATTTGGGGGAAAAATTGTTTDGGRTCAA The amount of HPV DNA
in the specimen was determined by semiquantitative method. Luminescence signal strengths in
relative light units (RLU) of the specimen were compared with standard positive controls
Observational Model: Cohort, Time Perspective: Prospective
Changes from baseline in viral loads of human papillomaviruses virus during and after target therapy
Study 1:The sampling of eyebrow hairs and skin scales before the initiation of biologics therapy, 12 and 24 weeks after initiation of the target therapies, 12 weeks after discontinuation of target therapies. Study 2: The same above mentioned microorganisms exams were done at enrollment and repeated again 12 weeks after the discontinuation of therapies. Comparison of the prevalence of latent virus and changes from baseline in viral load of human papillomaviruses virus between psoriasis patients who are treating with and without target therapies is performed.
before the initiation of biologics therapy, 12 and 24 weeks after initiation of the target therapies, 12 weeks after discontinuation of target therapies
Tsen-Fang Tsai, M.D.
National Taiwan University Hospital
Taiwan: Department of Health