Influenza Vaccination in Patients with Rheumatoid Arthritis under
treatment with TNFα Blockers or co-stimulus Inhibitor
Tutore
Prof. Raffaele D’Amelio
Coordinatore
Prof. Marco Tripodi
Dottoranda
Francesca Milanetti
Dottorato di Ricerca in Scienze Pasteuriane
XXV Ciclo
1
Table of Contents
1. Introduction
1.1 Vaccinations and Autoimmune Diseases
1.2 Could Autoimmunity Be Induced by Vaccination?
1.3 Are Anti-Infectious Vaccinations Safe and Effective in Patients with
Autoimmunity?
1.4 State of Art
1.5 Rheumatoid Arthritis
Influenza Vaccine
Pneumococcal Polysaccharide Vaccine
1.6 Systemic Lupus Erythematosus
Influenza Vaccine
2. Objective
3. Materials and Methods
3.1 Safety
2
3.2 Laboratory Evaluation
Specific anti-Influenza Antibodies
Specific anti-Pneumococcal Antibodies
Autoantibodies
Peripheral Blood Moninuclear Cells (PBMC)
Preparation of PBMC samples
Lymphocyte Subpopulations
PMA-iono stimulated samples
4. Statistical Analysis
5. Results
5.1 Safety
5.2 Immunogenicity
Anti-influenza antibodies
Anti-Pneumococcal Antibodies
5.3 Lymphocyte subpopulations
3
T-regulatory cells
PMA-iono stimulated PBMC
6. Discussion
7. References
4
1. Introduction
1.1 Vaccinations and Autoimmune Diseases
Vaccine administration is a very effective tool to prevent infectious diseases, but its
powerful stimulus on the immune system has generated the fear of exacerbating
previously diagnosed autoimmune diseases. Such a fear is also a consequence of the
generally anecdotal observation of post-vaccine autoimmune reactions in few
previously healthy susceptible individuals. Moreover, vaccine-related autoimmune
reactions have also been observed in animal models and extrapolated to man.
The possible role of vaccines in autoimmune disease induction is, however, difficult
to prove in humans, considering the need to perform large, complex, and expensive
epidemiological studies, thus limiting their availability [1]. Furthermore, patients
with autoimmune diseases have a higher risk, which may be estimated to be
approximately double that of the general population for developing infections,
mainly at respiratory level, as a consequence of a dysregulated immune system as
well as of immunosuppressive treatment [2].
In the last few years, in fact, the growing availability of even more powerful
immunosuppressive therapies, including biological agents, has allowed to better
control autoimmune diseases, but at the price of a more pronounced immune
suppression, which exposes patients to a higher infection risk [3], whose prevention,
via specific available vaccinations, is crucial. Nevertheless, vaccine coverage in
patients with autoimmune diseases is still relatively low [4–6], largely for lack of
recommendations by medical doctors [4, 5].
Sowden et al. could establish, in fact, that the percent of immunized patients with
rheumatic diseases under major immunosuppressive therapy but without comorbidities representing risk factors for infections, was significantly lower than the
percent of patients with co-morbidities (53% versus 95% and 28% versus 64% for
5
flu and pneumococcal vaccination, respectively), largely due to the less common
offer of vaccination by the primary care physicians [4]. In another study in the UK,
only 37% of patients with chronic inflammatory diseases under immunosuppressive
therapy had received both influenza and pneumococcal vaccinations [5], and the
situation is no different in France [6].
Thus, analyzing the problem, elaborating and releasing clear guidelines on the right
use of vaccines, and sensitizing the general population and healthcare workers on the
vaccine usefulness play a pivotal role in avoiding unfounded fears or presumed
contraindications that may keep a large part of the population away from
vaccinations, even in severe circumstances, like pandemic periods [7, 8].
In a recent issue of the International Reviews of Immunology, the matter has been
extensively analyzed by Salemi and D’Amelio grading the studies analyzed from I
to IV and from A to D the levels of evidence and the corresponding grades of
recommendation, respectively [9].
6
1.2 Could Autoimmunity Be Induced by Vaccination?
This hypothesis, formulated on the basis of generally anecdotal case reports, is
difficult to prove, due to the complexity to set up the needed larger and expensive
epidemiological studies. Moreover, the existing studies are generally not controlled,
but only observational, thus intrinsically having a rather low level of evidence, and
hardly comparable among them [10].
Although suggestive data have, therefore, been seldom reported, no conclusive
evidence has emerged [11].
Actually, autoantibody onset may occasionally follow vaccination, but activation of
the immunological regulatory mechanisms makes the transition to clinically overt
disease rare [1]. Only in a few conditions, the association between vaccine injection
and autoimmunity onset has reliably been either confirmed, such as in the case of
Guillain-Barr´e Syndrome after the 1976 swine influenza vaccine [1, 12–18] and of
immune thrombocytopenic purpura after measles/mumps/rubella (MMR)
vaccination [1, 12, 13, 19–21], or excluded, such as in the case of insulin-dependent
diabetes mellitus (IDDM) and type or timing of different vaccinations [22, 23].
Otherwise, the originally suspected association between hepatitis B vaccine and
multiple sclerosis (MS) [24, 25] has not been confirmed by larger studies [26–29],
even though it has been more recently reconsidered [30], also through further
observational studies [31, 32].
The large majority of experimental demonstrations has been performed either in
animal models or based on the proven molecular mimicry between
vaccine/infectious agents and host antigens. However, it should be underlined that
the experimental animal models, although of great speculative importance to infer
on the immunological mechanism, may hardly be directly transferred to the situation
of vaccines in humans and the only presence of shared epitopes is not sufficient per
se to induce disease. Areas of research still include the need to:
7
• set up larger and well constructed epidemiological studies on vaccine-related
autoimmunity onset, in order to obtain more reliable data;
• monitor autoimmune clinical and serological parameters during preregistration
studies [1];
• further clarify underlying immunological mechanisms; and
• perform genetic and proteomic systematic studies, in order to identify possible
predisposed individuals.
8
1.3 Are Anti-Infectious Vaccinations Safe and Effective in Patients with
Autoimmunity?
The effect of preventive vaccinations has been systematically analyzed up to now, in
over 5000 patients with MS, systemic lupus erythematosus (SLE), rheumatoid
arthritis (RA), IDDM, chronic arthritis in children, myasthenia gravis, or vasculitis.
Since the second half of the 1970s, the development of these studies is not casual;
only in 1976, in fact, following the swine influenza outbreak in the U.S., the USA
National Program for Flu Vaccination recommended to reassess risks and benefits of
flu vaccination in SLE [33].
In that period, the studies of preventive vaccinations in autoimmune diseases have
mainly been addressed to anti-influenza (also swine influenza vaccine) in patients
with MS [34–36], SLE [37–40], and RA [41]. In 1979, pneumococcal vaccination in
remitting or mild SLE patients was also demonstrated to be safe and immunogenic
[42].
Since the second half of the 1980s, IDDM has been extensively studied [43–45] and
at the end of the 1990s, for the first time, the contemporary administration of more
than one vaccine (pneumococcal polysaccharides, tetanus toxoid [TT], and
Haemophylus influenzae type b) in remitting SLE patients was demonstrated being
safe and immunogenic [46].
Only in the last decade did these studies show a net increase, mainly with regard to
anti-pneumococcal and influenza vaccinations in SLE and RA patients, the latter of
which were also under treatment with tumor necrosis factor (TNF)α blockers or
other biological agents.
Safety of vaccination has been demonstrated by the majority of publications,
provided that remitting or mild patients were chosen [47–49]; immunogenicity,
although protective, was generally lower than in normal controls.
Data are very poor on the efficacy. In particular, safety and immunogenicity of
influenza vaccination in MS, SLE, RA, and vasculitides and of pneumococcal
vaccination in SLE, immunogenicity of pneumococcal vaccination in RA, and
9
efficacy of influenza vaccination in MS have all been demonstrated through a high
level of evidence studies, thus reaching the highest grade of recommendation.
Preventive vaccinations should, therefore, be recommended [50] in patients with
low-activity autoimmune diseases.
To our knowledge, the large part of experience has until now been made with non
adjuvanted vaccines, considering that the adjuvanted vaccines (HB, TT) have only
been used in less than 20% of immunized patients. Thus, their safety should more
widely be evaluated and balanced toward the expected higher immunogenicity,
relevant in this group of immune compromised patients.
The immunosuppressive therapy in patients with autoimmune diseases has reached
considerable progress. The growing availability of drugs and biologicals, able to
very selectively hit the immune system, but also to induce broad functional
consequences, allows to more and more effectively treat the patients, but in parallel
exposes them to an increased infection risk.
The very nature of the disease and the heavy immune suppression cast doubts on
safety and immunogenicity of anti-infectious vaccinations in these patients.
Vaccination, however, is the only tool to prevent infections, thus improving the
prognosis of these patients.Until now, over 2500 patients affected by systemic lupus
erythematosus (SLE) and rheumatoiod arthritis (RA) have been systematically
studied (table 1). Safety and immunogenity of influenza and pneumococcal
vaccinations in RA and SLE patients are exposed in tables 2-3.
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Tab 1. - Cumulative Number of Vaccinated Patients with SLE and RA
Disease
Influenza vaccine
SLE
RA
522
996 (546)a
Total
1518
Pneumoccoccal
vaccine
305
710(-357)b
(396)a
Total
1015
2533
827
1706
SLE—systemic lupus erythematosus; RA—rheumatoid arthritis
aRheumatoid arthritis patients under treatment with biological agents and immunized with flu (415 with
TNFαblockers, 41 with Rituximab) or pneumococcal vaccine (328 with TNFα blockers, 68 with Rituximab).
bThree hundred fiftyseven patients have been vaccinated with both influenza and pneumococcal vaccines.
Tab 2. Clinical (No. of Patients Out of the Total with Post-Vaccine Clinical
Exacerbation) and Immunological (No. of Patients Out of the Total with PostVaccine New Onset or Rise in Titer of Auto-Antibodies) Safety of influenza and
pneumococcal vaccinations in RA and SLE patients
Disease
Vaccine
SLE
RA
Influenza
No. of
studies
18(1a16b)
No. of
pts
440
Pneumo
9(2a7b)
266
Influenza
11(1a7b)
423
Pneumo
1b
42
Clinical
safety
1/440
(3.86%)
2/266
(0,75%)
28/423
(6,61%)
Immunol
safety
33/212
(15,56%)
0/156
LoEc
Ref
IB
ND
IB
1/42
(2,38%)
ND
IIA
19-24, 2737, 39
40, 41, 4349
23,32,5862,64,66,6
8,71
47
IB
SLE—systemic lupus erythematosus; RA—rheumatoid arthritis
aRandomized, double-blind studies. bControlled studies. cLevel of evidence
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Tab 3. Immunogenicity (No. of Patients with Antibody Response to Vaccine
Antigens Out of the Total) of influenza and pneumococcal vaccinations in RA
and SLE patients
Disease
Vaccine
SLE
RA
Influenza
No.of
studies
12(1a11b)
No. of
pts
408
Pneumo
9(2a7b)
254
Influenza
11(2a9b)
779
Pneumo
8(1a7b)
702
Immunoge LoEc
nicityd
185/408
IB
(45,34%)
192/254
(75,59%)
386/779
(49,55%)
337/702
(48%)
IB
IB
IB
References
1924,28,31,32,36,3
8,39,40-47,49
40-47,49
23,32,58,6268,71
47,64,73-78
SLE—systemic lupus erythematosus; RA—rheumatoid arthritis
a
Randomized, double-blind studies. bControlled studies. cLevel of evidence. dImmunogenicity is
expressed for influenza as seroconversion rate (the lower between the 2 A vaccine antigens); for
pneumococcal vaccine as proportion of patients with at least twofold antibody increase toward at
least two polysaccharide antigens.
Areas of research still include the need to:
• evaluate vaccine safety also in patients with active disease;
• extend experience on safety and immunogenicity of adjuvanted vaccines;
• evaluate safety and immunogenicity of not yet explored vaccines or adjuvants.
Finally, a deeper knowledge of vaccine-induced in vivo immune system modulation
should also be considered.
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1.4 State of Art
Several publications have reported generally anecdotal cases of autoimmune
reactions after preventive anti-infectious vaccinations. Most of the available
publications refer to case-reports and observational studies.
Mechanisms through which vaccines may induce autoimmune diseases are mainly
extrapolated by the known capacity of infectious agents against which vaccines are
directed to induce autoimmunity, and by animal models and in vitro studies [51].
An attempt has been made to organize the topic of post-vaccine autoimmunity onset
according to the proven or suggested immunological mechanisms, which may be
antigen-specific (molecular mimicry) or non specific (bystander activation) [52–54].
Molecular mimicry is based on the structural similarity between micro-organisms
and host antigens, such as either the epitopes recognized by anti-group A βhaemolytic Streptococcus antibodies cross reacting with heart tissue host antigens in
rheumatic fever [55] or the produced monoclonal antibodies to measles and
herpesviruses cross reacting with self proteins [56].
Molecular mimicry may also be identified at T-cell level, like in the model of
experimental autoimmune encephalomyelitis (EAE) induced in rabbits through the
injection of a hepatitis B (HB) polymerase peptide mimicking myelin basic protein
(MBP) in complete Freund’s adjuvant (CFA) [57].
Another example is the herpetic stromal keratitis induced by the herpes simplex
virus 1 (HSV-1) [52]. It has, in fact, been demonstrated in mice that T helper (Th)1
clones, elicited by HSV-1, were able to cross recognize an HSV-1 peptide and self
antigens, such as a corneal protein and immunoglobulin (Ig) G2a.
It should be underlined that the only presence of shared epitopes is not sufficient per
se to induce disease, but it is necessary to have mimicry with a peptide that may be
presented to autoreactive T-cell clones [54]. It should, however, be remembered that
T cell recognition is characterized by a high level of polyspecificity, according to the
Mason’s calculation on the theoretical possibility for one T cell receptor to
recognize more than one million different linear epitopes [58].
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Finally, for an autoimmune disease to occur, in addition to the molecular mimicry,
the contemporary presence of an infection or at least of a strong adjuvant, such as
CFA, is necessary [55].
Bystander activation is mainly inferred from studies of experimental animal models
and it is based on the release of sequestered self antigens from the infected host
tissue, leading to activation of antigenpresenting cells, able to stimulate preprimed
dormant autoreactive T-cell clones [53]. Alternatively, virus-specific T-cells may
initiate the process by killing the virus-infected cells aftermigrating to the affected
organ, thus releasing self antigen and contributing, with macrophages, to the local
high cytokine levels and consequent inflammation [53]. Examples are the
encephalomyelitis induced in mice by Theiler’s murine encephalomyelitis virus or
type 1 diabetes induced by Coxsackie B4 virus [9, 10] in mice having high levels of
islet-antigen specific T-cells.
In addition to the above-reported examples, these mechanisms have been suggested
or demonstrated to be involved in several additional infection- or vaccine-induced
autoimmune diseases, such as treatment-resistant Lyme arthritis and Borrelia
burgdorferi sensu stricto [61], multiple sclerosis (MS) and hepatitis B (HB) vaccine
[62,63], Guillain-Barr´e Syndrome (GBS) and Campilobacter jejunii [64], or swine
influenza vaccine [65]. These models represent the scientific basis for also
interpreting possible post-vaccine autoimmune reactions and provide data in order to
avoid molecular mimicry in the construction of new vaccines.
This may be the case for group A β-haemolytic streptococcal vaccine or vaccine
against Neisseria meningitides serogroup B. It should be underlined that animal
models may hardly be directly transferred to humans. Moreover, the presence of
auto-reactive T-cell clones or the identification of auto-antibodies does not mean
that an autoimmune disease will automatically develop following infection or
vaccination. For such an event to occur, in fact, it is necessary to satisfy additional
pre-conditions, including presence of stimulating cytokines in order to activate a
critical mass of autoreactive clones as well as lack of effective regulatory
mechanisms [53].
14
Thus, the actual prevalence of autoimmune reactions to vaccination is remarkably
low (much less than 1:10,000 of the hundreds of millions vaccine doses yearly
administered in the world [66, 67]), probably as a consequence of the many
redundant regulatory mechanisms active in the immune system [53]. Nevertheless,
the experimental models may provide a biological plausibility [51] to the hypothesis
of a link between vaccine administration and autoimmune disease onset, generally
formulated on the basis of single case-reports. Only in a few conditions has such an
association been more reliably confirmed on the basis of larger observational studies.
However, for the majority of the hypothesized associations, larger, complex, and
expensive epidemiological studies should be needed, but they are still lacking for the
difficulty to be performed [53].
Moreover, a vaccine-related autoimmune reaction should meet a series of
requirements to be actually considered vaccine-induced [68], including consistency,
strength, specificity, and temporal relation. In particular, the reaction should occur in
different populations and be observed by different physicians, with a strong
epidemiological association, only linked to a specific type of vaccine and within a
time-period of a few weeks following vaccination [53].
Recently, however, temporal relationship between either infection or vaccination
and autoimmunity onset has been challenged, by suggesting a more extensive period
involving months/years [69]. Comparing infection- versus vaccine-induced
autoimmune reactions, the latter generally show a lower incidence, with the majority
of them displaying a milder and self-limiting clinical course [67]. These
observations are in agreement with a favorable cost-effectiveness ratio of vaccine
use. New onset of auto-antibodies not accompanied by any clinical disease after
different vaccine administration has been described.
Already at the beginning of the 1960s, a transient rise of rheumatoid factor (RF)
after immunization of healthy people or rheumatoid arthritis (RA) patients with a
variety of vaccines, including tetanus toxoid (TT), typhoid-paratyphoid A and B
(TAB), diphtheria, polio, smallpox, and mumps [70, 71] had, in fact, been reported.
The inactivated TAB parenteral vaccine, but not the living oral typhoid vaccine, was
15
able to induce a transient IgM RF positivity 15 and 30 days after vaccination, with
return to baseline values by 8 months, not associated with any clinically apparent
autoimmune disease [72].
An inactivated vaccine against Coxiella burnetii, the etiological agent of Q fever,
was able to induce in recipients the onset of anti-Ig antibodies (anti-Fab [40%], antiIgA [20%], and anti-IgG and IgM [15%]), not accompanied by any clinical sign [73].
Recently, Toplak et al. observed an increase or appearance of auto-antibodies in 15
and 13% of 92 apparently healthy medical workers (with a baseline high rate of
auto-antibody positivity), 1 and 6 months after flu vaccination, respectively,
suggesting de novo induction of auto-antibodies after influenza vaccination in
selected individuals [74].
Clinical syndromes described after vaccination mainly include neurological pictures
represented by MS, acute disseminated encephalomyelitis (ADEM), optic neuritis
(ON), transverse myelitis (TM), GBS, and brachial neuritis (BN). Immune
thrombocytopenic purpura (ITP), rheumatic syndromes, such as RA, systemic lupus
erythematosus (SLE), vasculitis, reactive arthritis, Sjogren’s syndrome and
inflammatory myopathies (IM), myopericarditis, or finally, insulin- dependent
diabetes mellitus (IDDM) have also been described [67].
Among the several reports of autoimmune diseases whose onset has been attributed
to vaccinations in predisposed individuals, those reliably vaccine-associated may be
considered GBS after 1976 swine influenza vaccine, ITP after MMR vaccination [53,
88, 89], and myopericarditis after smallpox vaccination [88], whereas the suspected
association between HB vaccine and MS [62, 63] has not been subsequently
confirmed [80–82], even though it has been recently reconsidered [82–85], and the
one between childhood immunization and IDDM seems, by now, to be definitively
reduced [67, 86, 87].
However, the need to perform larger and well-constructed epidemiological studies
on vaccine-related autoimmunity onset, in order to obtain more reliable data, should
be underlined. In conclusion, the relationship between vaccinations and autoimmune
diseases should be considered in a complex and bidirectional way; vaccinations, in
16
fact, prevent infections, which may in turn trigger autoimmunity, but, on the other
hand, vaccinations themselves, by a specific mechanism of molecular mimicry, or a
non specific mechanism of bystander activation or finally by yet unknown
mechanisms, may trigger autoimmunity [90].
However, although vaccine administration has been associated with autoimmune
manifestations in selected predisposed individuals, who may not be currently
identified [53], their very low incidence and mild clinical course, generally lower
and milder than those associated with natural infections [67], combined with the
general lack of high level of evidence, should not induce to abstain from the
immunization practice considering the favorable cost-effectiveness ratio of vaccine
use.
However, areas of research still include the need to develop autoimmune clinical and
serological monitoring during preregistration studies [53], perform genetic and
proteomic systematic studies in order to identify possible predisposed individuals,
and finally, further clarify underlying immunological mechanisms exerted by
vaccines/adjuvants.
17
1.5 Rheumatoid Arthritis
Influenza Vaccine
In 1979, in a controlled study on 17 RA patients treated with influenza bivalent
vaccine (A/New Jersey/76 and A/Victoria/75), six of them developed a slight
disease reactivation within 3 weeks from vaccination, a figure in line with the
spontaneous rate of flare-ups. Moreover, vaccine immunogenicity was comparable
between the two groups of RA patients and normal controls. In particular, 3 weeks
after immunization 11/17 (65%) RA patients presented a fourfold antibody titer
increase towards both vaccine antigens [93].
In 1988, Turner Stokes et al. observed an impaired in vitro antibody response to
influenza vaccine antigens in 2/10 immunized RA patients [94].
In 1994, Chalmers proposed including RA patients in the standard immunization
programs, on the basis of a randomized double-blind, placebo-controlled study on
126 RA patients, half of whom were vac- cinated for influenza. The rate of flare-ups
was the same in both vaccinated and non-vaccinated RA patients, and also the rate
of an- tibody titer increase was comparable among the 63 vaccinated RA pa- tients
and 61 vaccinated healthy controls. Regarding immunogenicity, seroconversion rate
in RA patients was 77% for A/H3, 50% for A/H1, and 37% for B antigen [128].
In 1994, Cimmino and colleagues performed a retrospective study during a fourmonth period based on interviews of all consecutive RA outpatients about previous
influenza vaccination and disease flares in the month following immunization.
Thirty out of 123 evaluated patients had received at least one influenza vaccination
during their lifetime and 23/123 had been vaccinated during the year of the study.
RA flare was reported in 6/30 patients after vaccination, a rate similar to the
expected rate of spontaneous exacerbation [129].
In 1996, Francioni et al. could demonstrate safety and satisfying immunogenicity of
influenza vaccination by an open study including 40 RA patients divided into two
groups (26 in remission and 14 with active disease) compared to healthy controls, all
18
immunized during the 1991–1992 winter season. Regarding safety, they observed
disease worsening in 8/40 RA patients. The antibody response was adequate to
provide protection from the infection in 27/40 RA patients [130].
In 2003, Iyngkaran et al. reported severe vasculitis onset in a young female RA
patient 2 weeks after flu vaccination with an inactivated trivalent vaccine [131].
Since 2006, different studies on flu vaccination in RA patients under treatment with
both Disease-Modifying Anti-Rheumatic Drugs (DMARDs) and tumour necrosis
factor (TNF)α blockers have been pub- lished. In particular, Fomin et al. in 82 RA
patients under treatment with DMARDs (either MTX alone or MTX and PDN) and
TNFα blockers (22/82 with Infliximab [INF] and 5/82 with Etanercept [ETN]) and
30 age- and sex-matched healthy controls, did not observe any significant RA
clinical or laboratory worsening after flu vaccination with a split in- activated
vaccine without adjuvant. Moreover, vaccine immunogenicity was adequate 6 weeks
after vaccination, considering that seroconver- sion rate was 67% for B antigen and
53% for A/H3 and A/H1 [132].
Del Porto et al. analyzed 10 RA patients with low-moderate (DAS28 < 4) and stable
disease activity, two of whom were under treatment with TNFα blockers, before and
after vaccination with a flu inactivated split vaccine without adjuvant [102]. Ten RA
non-vaccinated patients and ten vaccinated healthy people were considered as
controls. All pa- tients (vaccinated and non) were treated with PDN <10 mg/daily
plus either MTX (10 mg/weekly) or cyclosporine A (CyA <3 mg/kg/daily). No
significant difference between vaccinated and non-vaccinated RA patients was
observed regarding DAS28 modifications and flare-up inci- dence (2/10 vaccinated
versus 3/10 non-vaccinated control RA patients). Furthermore, lymphocyte
subpopulations, including regulatory T cells, did not show any significant
modification in RA patients, compared with controls, 1, 3, and 6 months after
vaccination. Finally, specific antibody response toward flu vaccine antigens was
quite a bit lower in patients than healthy controls. In particular, seroconversion rate
was 40% for A/H1, 50% for A/H3, and 20% for B antigen. No RA patient developed
influenza during the 6-month follow-up. Stojanovich could demonstrate safety of
19
trivalent flu vaccine in 23 RA patients in sta- ble condition longitudinally observed
together with 31 non-vaccinated RA control patients; the vaccine was, in fact, well
tolerated and the number of respiratory infections was significantly lower in
vaccinated patients. In particular, 0/23 versus 0/31 cases of pneumonia, 1/23 ver- sus
7/31 cases of acute bronchitis, and 2/23 versus 19/31 cases of viral respiratory
infections, respectively, were observed [104].
In 2007, Kapetanovich et al. studied 149 RA patients (62 treated with TNFα
blockers alone or combined with DMARDs other than MTX, 50 with TNFα
blockers and MTX, and 37 with MTX alone) and 18 healthy subjects,
simultaneously vaccinated with trivalent inactivated flu and pneumococcal
polysaccharide vaccines. Seroconversion rate of unprotected RA patients at baseline
was 60.58% for A/H1, 60.83% for A/H3, and 81.63% for B antigen. Lower flu
antibody response was ob- served in RA patients under treatment with TNFα
blockers alone or combined with DMARDs [133].
In a randomized, double-blind, placebo- controlled, multicenter study, Kaine et al. in
the same year, analyzed 208 RA patients treated with either MTX and placebo or
MTX and Adal- imumab (ADA) and who were simultaneously vaccinated with
trivalent influenza and pneumococcal polysaccharide vaccines. Four weeks after
vaccination, specific antibody response toward flu vaccine antigens was lower in RA
patients under ADA. In fact, the seroconversion rates in the two groups were 56%
versus 50.5% for A/H1, 67.9% versus 58.6% for A/H3, and 60.6% versus 48.5% for
B antigen, respectively. However, if the antibody levels at baseline were considered,
the patients with protective titers showed antibody response after vaccination
compara- ble with that of RA patient group receiving placebo [134].
Kubota et al. in 27 RA patients under treatment with TNFα blockers (11 with INF
and 16 with ETN), 36 under treatment with DMARDs alone, and 52 healthy controls
all vaccinated with trivalent flu vaccine, observed a higher seroconversion rate in the
patient group under treatment with TNFα blockers. In particular, in the group under
treatment with TNFα blockers, the seroconversion rate was 44.44% for A/H1 and
A/H3 and 26.63% for B antigen, whereas in the other group the corresponding
20
values were 22.22%, 33.33%, and 22.22% [135].
In a prospective cohort study in 2005, 79 RA patients (52 under treatment with
TNFα blockers and 27 with DMARDs) and 18 healthy controls showed adequate
antibody response four weeks after flu vac- cination with a subunit vaccine, although
antibody titers and serocon- version rate were lower in RA patients treated with
TNFα blockers. In particular, in this group the seroconversion rate was 50% for
A/H3, 46.15% for A/H1 and B, whereas in the group under DMARDs the corresponding values were 44.44%, 51.85%, and 66.6% [136].
Elkayam and colleagues performed a study on 43 RA patients and 17 healthy
controls, immunized with split flu vaccine contain- ing A/NewCaledonia/20/1999,
A/Wisconsin/67/2005, and B/Malaysia/ 2506/2004 antigens. Twenty RA patients
were under treatment with INF, while 23 RA patients were only treated with
DMARDs. INF- treated patients were further subdivided into two groups: 13 were
vac- cinated the same day of INF administration, while seven received the vaccine 3
weeks later. No significant modifications in disease activity have been observed as a
consequence of vaccination, but only a slight increase of erythrocyte sedimentation
rate 6 weeks after vaccination. In all the explored groups, antibody titer toward the
three vaccine antigens increased, except in RA patients immunized 3 weeks after
INF administration, in whom a rise of antibody titer only against the B/Malaysia
antigen was demonstrated. Moreover, seroconversion rate was 45% for A/H1, 60%
for A/H3, and 44% for B vaccine antigens [137].
In 2009, Salemi et al. in 28 RA patients under treatment with TNFα blockers
immunized with influenza vaccine, could confirm vac- cine safety and
immunogenicity, but they were also able to demon- strate specific antibody titer
persistence at protective levels 6 months after vaccination and strengthened the
importance of annual vacci- nation, considering that in a cohort of 18 patients
vaccinated each year for 3 consecutive years, the low antibody response to B
vaccine antigen could be overcome. Moreover, a significant T regulatory cell
increase was observed, only in RA patients, 1 month after vaccina- tion [138].
In the same year Nii et al. [139] could demonstrate in 51 RA patients (26 of whom
21
under treatment with TNFα blockers) immu- nized with influenza vaccine and reevaluated for specific antibodies after 1 year, that protective antibody levels were
still maintained by 30.8 and 52.0% of RA patients under treatment with biologicals
or not, respectively, versus 46.4% of healthy controls, not a significant difference.
Regarding treatment with biologicals different from TNFα block- ers, the response
of RA patients treated with anti-B lymphocyte monoclonal antibodies (rituximab
[RTX]) to influenza vaccination has been reported in three papers [140–142].
First, Gelinck et al. in 2007, observed a lower specific antibody response to flu
vaccination in four RA patients under treatment with RTX and MTX than in 19 RA
pa- tients under treatment with TNFα blockers with or without DMARDs and 20
healthy controls, all matched for sex, age, and antibody titer at baseline, 84 days
after the first RTX administration. From the pre- sented data, seroconversion rate
could not be argued [140].
The year after Oren et al. evaluated the response to flu vaccine in three groups of
subjects (14 RA patients treated with RTX, 29 RA patients treated with DMARDs,
and 21 healthy controls). After vaccination, no dis- ease reactivation was observed.
Moreover, the seroconversion rate for the A/H3 antigen was significantly lower in
RA patients under RTX than in controls (21% versus 67%), whereas no significant
difference could be observed in relation to the other vaccine antigens (38% ver- sus
37% for A/H1 and 21% versus 30% for B) [141].
In 2010, van Assen confirmed this data on 23 RA patients under RTX, 11 of whom
were immunized against influenza 4 to 8 weeks from RTX adminis- tration and 12
after 6 to 10 months, 20 RA patients under MTX, and 29 healthy controls. RTX, in
fact, markedly reduced specific antibody response compared to controls, with a
slightly restored response 6 to 10 months after RTX administration. From the
presented data, sero- conversion rate could not be argued. Regarding safety, DAS28
was not influenced by flu immunization [142].
In 2010 in a controlled trial, Bingham [143] enrolled 103 patients with active RA
receiving a stable dose of methotrexate (MTX). Tetanus toxoid, pneumococcal
polysaccharide, and KLH vaccines as well as a Candida albicans skin test were
22
administered to 1 group of patients receiving rituximab plus MTX (called rituximabtreated patients) for 36 weeks and to 1 group of patients receiving MTX alone for 12
weeks. In this study Rituximab-treated patients had decreased responses to
pneumococcal polysaccharide vaccine (57% of patients had a 2-fold rise in titer in
response to >or=1 serotype, compared with 82% of patients treated with MTX
alone).
In 2011 Arad [144] administrated trivalent influenza subunit vaccine to 46 RA
patients and to 16 healthy controls. They showed that cell-mediated responses were
comparable in RTX-treated vs. DMARDs-treated patients. The recall
postvaccination CD4+ cellular response was similar in RA patients and healthy
controls. A positive correlation was found between CD19+ cell count on the day of
vaccination and cellular response in RTX-treated RA patients The antibody response
rate was significantly impaired in the RTX group: being 26.4%, 68.4% and 47.1% in
RTX-treated, DMARDs-treated and controls, respectively.
Elkayam O et Al [145]. showed that vaccination against pandemic H1N1 using an
adjuvanted H1N1v monovalent influenza was safe and induced an appropriate
response in patients with RA and SLE.
Saad CG in 2011 [146] studied 1668 ARD patients (systemic lupus erythematosus
(SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), systemic sclerosis,
psoriatic arthritis (PsA), Behçet's disease (BD), mixed connective tissue disease,
primary antiphospholipid syndrome (PAPS), dermatomyositis (DM), primary
Sjögren's syndrome, Takayasu's arteritis, polymyositis and Granulomatosis with
polyangiitis (Wegener's) (GPA)) and 234 healthy controls that were vaccinated with
a non-adjuvanted influenza A/California/7/2009(H1N1) virus-like strain flu. After
immunization, seroprotection rates, seroconversion rates and the factor increase in
GMT were significantly lower in ARD than controls. Analysis of specific diseases
revealed that seroprotection significantly reduced in SLE, RA, PsA, AS, BD and
DM patients than controls. The seroconversion rates in SLE, RA and PsA patients
and the increase in GMTs in SLE , RA and PsA patients were also reduced
compared with controls.
23
In a Ribeiro [147] study 340 adult RA patients and 234 healthy controls were
assessed before and 21 days after adjuvant-free influenza A/California/7/2009
(pH1N1) vaccine. RA and controls showed similar prevaccination GMT and
seroprotection (10.8% vs 11.5%). After vaccination a significant reduction was
observed in all endpoints: GMT and factor increase in GMT, seroprotection and
seroconversion rates. Disease activity did not preclude seroconversion or
seroprotection and remained unchanged in 97.4% of patients. Methotrexate was the
only disease-modifying antirheumatic drug associated with reduced responses.
Kobie [148] showed a decreased influenza-specific B cell responses in rheumatoid
arthritis patients treated with anti-tumor necrosis factor. Compared with healthy
controls, RA patients treated with anti-TNF exhibited significantly decreased
influenza-specific serum antibody and memory B cell responses throughout multiple
years of the study. The short-term influenza-specific effector B cell response was
also significantly decreased in RA patients treated with anti-TNF as compared with
healthy controls, and correlated with decreased influenza-specific memory B cells
and serum antibody present at one month following vaccination.
In 2012 Mori [149] studied the effect of Tolicizumab (TCZ) therapy on antibody
response to influenza vaccine in patients with rheumatoid arthritis. In this study TCZ
does not hamper antibody response to influenza vaccine in RA patients. Influenza
vaccination is considered effective in protecting RA patients receiving TCZ therapy
with or without MTX.
Ribeiro [150] demonstrated that Abatacept severely reduces the immune response to
pandemic 2009 influenza A/H1N1 vaccination in patients with rheumatoid arthritis.
In another study, Milanovic [151] shown the efficiency, sufficient immunogenicity
and safety of modern influenza vaccine application in patients suffering from
systemic lupus erythematosus, rheumatoid arthritis or Sjögren's syndrome.
França IL [152] compared one hundred and twenty patients (RA, n = 41; AS, n =
57; PsA, n = 22) on anti-TNF agents (monoclonal, n = 94; soluble receptor, n = 26)
with 116 inflammatory arthritis patients under DMARDs and 117 healthy controls.
They observed no differences in RA patients receiving anti TNF-alpha therapy
24
compared with healthy controls.
In conclusion, despite the evidence coming from only one random-ized and seven
controlled but non-randomized studies, inactivated flu vaccine seems generally safe
in 996 RA patients (Table I), 540 of whom are under treatment with only DMARDs,
the remaining are under treatment with biologicals, 456 with TNFα blockers and 41
with RTX. Immunogenicity in patients treated with DMARDs was generally comparable to that observed in normal controls, controversial in patients treated with
TNFα blockers, and significantly lower in patients under RTX.
Pneumococcal Polysaccharide Vaccine
In 1996, O’Dell et al. studied 40 RA patients before and after pneumococcal
polysaccharide vaccine administration; specific antibody response reached
protection level in 80% of them 6 weeks after vacci- nation. Response levels were
lower in patients under MTX, whereas age, sex, and use of other
immunosuppressive agents, such as steroids, AZA, sulfasalazine (SSZ), and HCQ
did not appear to be able to influ- ence antibody response [153].
In 2002, Elkayam et al. demonstrated vaccine safety and immuno- genicity in 42 RA
patients and 20 healthy controls immunized with a 23-valent pneumococcal
polysaccharide vaccine. Two months after vaccine administration the number of
tender joints was significantly lower than the number observed before vaccination.
Only one patient suffered a transient arthralgia and myalgia the day after vaccine administration. Specific immune response toward seven vaccine antigens 1 month after
vaccine administration showed a significant rise of GMT, but with profound
differences among the various antigenic serotypes. Moreover, positive response
(either at least twofold increase of anti- body titer or reaching serum antibody level
of 1 μg/ml) in the patient group was only obtained by 34 to 71% of patients toward
the differ- ent antigens, compared with the major percentage observed in healthy
controls. Finally, 1/3 of the patients did show either no response to polysaccharide
antigens or only to a single antigen and such a poor response was not related to the
25
vitamin B12 or pholate levels nor to the use of immunosuppressive agents, including
steroids, MTX, HCQ, or AZA [117].
In 2004, the same group performed a comparative study between 11 RA patients
under treatment with DMARDs and TNFα blockers (INF or ETN) and 17 RA
patients under treatment with only DMARDs, matched for age, sex, length of
disease, and use of MTX, before and after a 23-valent pneumococcal polysaccharide
vaccine administration. GMT of specific antibodies towards seven of the antigens
included in the vaccine 1 month after immunization showed a statistically significant increase in both groups, even if at a lower level, excepting serotype 14, for the
group under treatment with biological agents. Moreover, a significant lower
percentage (69% versus 82%) of patients under treat- ment with biological agents
showed a positive response toward two or more tested antigens [154].
On the contrary, Kapetanovich et al. in a larger study, were able to demonstrate that
MTX treatment could negatively influence the response to pneumococcal vaccine. In
fact, they studied 149 RA pa- tients (62 under treatment with INF or ETN together
with a DMARD other than MTX, 50 under treatment with INF or ETN associated to
MTX, and 37 with MTX alone). The percentage of patients with antibody response
to the antigens 23F and 6B was 50% in the group under treatment with biological
agents without MTX, 32% in the group under treatment with TNFα blockers with
MTX, and 13% in the group under treatment with MTX alone [155].
Similarly, Visvanathan et al. by analyzing specific antibody response to
pneumococcal polysaccharide vaccine in 70 patients with early RA (20 under
treatment with INF 3 mg/kg and MTX, 36 with INF 6 mg/kg and MTX, and 14 with
placebo and MTX), could conclude that adding INF to MTX did not influence the
response to vaccine, evaluated as a twofold increase in the antibody titer toward 12
tested vaccine antigens. In particular, the proportion of patients responding to two or
more tested antigens was 64% in the patient group treated with INF 3 mg/kg and
MTX, 70% in the group with INF 6 mg/kg and MTX, and 72% in the group under
treatment with MTX alone [156].
Kaine et al. through a multicenter, double-blind study on 208 RA patients under
26
DMARDs, randomized to receive ADA or placebo, simultaneously vaccinated with
23-valent pneumococcal polysaccharide and trivalent flu vaccines, could
demonstrate that the response to pneumococcal vaccine (twofold increase of
antibody titer towards at least 3/5 tested antigens) four weeks after vaccination was
similar in both groups (37% for ADA-treated versus 40% for placebo), as well as the
patient percentage reaching a protective antibody titer (placebo 81.7% versus ADA
85.9%) [134].
More recently, Gelinck et al. on 45 RA patients under treatment with DMARDs and
TNFα blockers, 28 RA patients treated with only DMARDs, and 18 healthy controls,
all immunized with pneumococcal polysaccharide 23-valent vaccine, could observe
an antibody response (defined as the percent of subjects with an antibody titer >0.35
μg/ml in addition to at least a twofold increase of antibody titer) toward four
polysaccharide vaccine antigens (6B, 9V, 19F, 23F) reduced in RA pa- tients under
MTX and even more in RA patients under MTX and TNFα blockers. In particular,
9/24 (36%) patients under only MTX, 4/27 (18%) patients under MTX + TNFα
blockers, 6/9 (67%) patients under only TNFα blockers, and 9/13 (70%) of patients
without both MTX and TNFα blockers were able to respond according to the above
reported parame- ters [157].
In 2010, Bingham et al. analyzed 100 RA patients, 32 of whom under MTX and the
remaining under MTX + RTX, immunized with TT and 23-valent pneumococcal
polysaccharide vaccine. The response to the T-independent pneumococcal
polysaccharide vaccine was markedly reduced in the RTX-treated patient group
[27/63 (43%) had ≥ twofold antibody rise towards at least two serotypes compared
to 22/28 (82%) observed in RA patients treated with only MTX] [158].
Also, in 32 patients with Sjo ̈gren’s syndrome, 14-valent pneumococcal
polysaccharide vaccine administration was demonstrated as being safe (no clinical
or serological disease exacerbation has been observed) and immunogenic
(persistence of antibody levels above baseline for 8/14 serotype antigens six months
after immunization) already in 1980 through a double-blind, placebo-controlled
study [159].
27
Coulson [160] evaluated Pneumococcal antibody levels after pneumovax in patients
with rheumatoid arthritis on methotrexate. They analyzed 152 patients, of whom 28
had never been vaccinated against pneumococcus. Median levels of antibodys were
significantly higher in those who had been vaccinated. Unvaccinated patients and
those taking oral prednisone were more likely to have had pneumonia in the
previous 10 years. The RR for developing pneumonia among non-vaccinated
patients was 9.7 (p=0.005) and among steroid-treated patients was 6.5 (p=0.001),
after adjusting for age, gender, disease duration and comorbidity.
Kapetanovic et al. analyzing 505 pz concluded that antibody response is reduced
following vaccination with 7-valent conjugate pneumococcal vaccine in adult
methotrexate-treated patients with established arthritis, but not those treated with
tumor necrosis factor inhibitors [161].
In another study 253 RA patients were studied: heptavalent pneumococcal
conjugate vaccine elicits similar antibody response as standard 23-valent
polysaccharide vaccine in adult patients with RA treated with immunomodulating
drugs [162].
An analysis of 398 patients show Persistence of antibody response 1.5 years after
vaccination using 7-valent pneumococcal conjugate vaccine in patients with arthritis
treated with different anti-rheumatic drugs [163].
In conclusion, the T-independent pneumococcal polysaccharide vac- cine, until now
performed on 710 RA patients (Table I), 328 of whom under TNFα blockers and 68
under RTX, has been shown to be safe; the immunogenicity is heavily reduced by
RTX and generally seems to be more compromised by MTX than by TNFα blockers.
However, although it should be better to start therapy after vaccination in order to
reach optimal antibody response, pneumococcal vaccine administration should be
recommended in these patients, even if already under treatment.
28
1.6 Systemic Lupus Erythematosus
Influenza Vaccine
In 1978–1979, the problem of vaccine administration in SLE patients has been faced
in a series of studies [164–168] all addressed to influenza prevention by swine
monovalent vaccine antigen A/New Jersey/76 or bi- valent A/New Jersey/76 and
A/Victoria/75. These studies demonstrated substantial vaccination safety, despite
two cases of glomerulonephritis arising in two patients with active SLE before
immunization [166, 167].
Moreover, these studies were able to demonstrate an immunogenicity comparable to
that of normal controls, except for two papers where SLE patients presented specific
antibody titers, after flu immunization with a bivalent inactivated vaccine, lower
than the ones observed in age- and sex-matched normal controls [164, 167].
In particular, Ristow et al. by vaccinating 29 SLE patients with an A/New Jersey/76
influenza vaccine, observed a fourfold increase of specific antibody in 14/29 patients,
whereas Williams et al. in 6/19, Brodman et al. in 13/46, and Louie et al. in 7/11.
The year after, Herron et al. analyzed 20 SLE patients before and after immunization
with a bivalent influenza vaccine and could observe four mild flare-ups, whereas the
antibody titer was not dissimilar from that observed in normal controls. Finally, the
serocon- version rate was 81% for A/New Jersey/76 and 67% for A/Victoria/75
[168].
In 1988 Turner Stokes and colleagues reported an impaired in vitro response in 9/28
SLE patients, in correlation with dsDNA auto- antibody levels and independently of
disease activity or immunosup- pressive therapy. In the in vitro non-responder group,
an in vivo seroconversion could, however, be observed in 23/28 patients, proba- bly
related to lymphoid redistribution of antibody-producing cells, as argued by parallel
lymph-node analysis [169, 170].
Between 2000 and 2002, Abu-Shakra et al. in 24 SLE patients treated with trivalent
split flu vaccine, analyzed possible disease re- activation [171], possible
29
appearance/increase of auto-antibodies [172], and specific anti-influenza antibodies
[173]. Disease activity was moni- tored by the SLE Disease Activity Index
(SLEDAI), a validated tool for disease activity monitoring [174]; no modification
was observed 6 and 12 weeks after vaccination [171]. A transient increase of autoantibodies (anti-Sm, anti-RNP, anti-Ro, anti-cardiolipin) has been observed at 6
weeks, independently of clinical disease activity [172]. Finally, specific antiinfluenza antibody response was lower than the one observed in the general
population (mainly in <50 years, under treatment with prednisone [PDN] >10 mg
per day or azathioprine [AZA], but not methotrexate [MTX]), with a seroconversion
rate of the total group of 58% for A/Sidney/97, 62.5% for B/Harbin/94, and 37.5%
for A/Bejing/95 [173].
In 2004, Mercado et al. reported 18 female SLE patients immu- nized with
inactivated flu vaccine and could observe absence of disease reactivation with a
protective immunogenicity, even though at lower levels than in healthy controls. In
particular, the seroprotection rate (the percent of patients with a geometric mean titer
of at least 1:40) was 67%, 72%, and 61% against A/Moskow/10/99, A/New
Caledonia 20/99, B/Sichuan/379/99 in SLE patients versus 77%, 94%, and 94%, in
healthy controls. The seroconversion rate could not be argued from data presented in
the paper [175].
In 2006, Holvast et al. [176] and Del Porto et al. [177] described 56 and 14 SLE
patients respectively, treated with trivalent split influenza vaccines in the 2003/2004
winter season. In both papers, vaccine safety was observed, provided that quiescent
SLE patients were chosen. Regarding immunogenicity, specific antibody titers
observed in SLE patients were not significantly different compared to 18 age- and
sex-matched healthy controls in the Holvast et al. experience, excepting the
seroconversion rate, which was significantly lower than the same parameter
observed in normal controls (A/H1 43%, 24/56; A/H3 39%, 22/56; and B 41%,
23/56 versus A/H1 94%, 16/17; A/H3 88%, 15/17; and B 71%, 12/17), this
reduction being related to AZA use [176]. Also, Del Porto et al. were able to observe
a protective antibody response against influenza virus and a seroconversion rate of
30
57%, 93%, and 36% for A/Moskow/10/99, A/New Caledonia 20/99, and
B/Shangdong/7/97 versus 70%, 50%, and 50% in healthy controls. The vaccine
efficacy was clinically evaluated by monitoring three ILI cases observed during the
seasonal period, only one of which was actually confirmed by viral identification.
Moreover, no increase/appearance of auto-antibodies was observed after flu vaccination, whereas 2/14 flare-ups have been observed after vaccination versus 1/10 in
non-immunized SLE patients [177].
In the same year, Tarja` n et al. analyzed the appearance/increase of anti-cardiolipin
anti- bodies in 18 low activity, stable SLE patients (8 of whom with a positive
history for anti-cardiolipin and/or β2-glycoprotein 1 antibodies) after vaccination
with trivalent flu vaccine for three consecutive years. Anti- β2 glycoprotein 1
antibodies increased in 7/8 patients with antibody positivity at baseline, whereas no
antibody appearance was observed in ten negative patients at baseline. Moreover, a
significant increase of anti-dsDNA antibodies was observed, mainly in the group
with base- line positivity of anti-cardiolipin antibodies. However, such immunological modifications were not accompanied by SLEDAI variations nor by
thrombotic events [178].
Again, in the same year, Stojanovich could demonstrate safety of trivalent flu
vaccine in 23 SLE patients in sta- ble condition longitudinally observed together
with 46 non-vaccinated SLE patients; the vaccine was, in fact, well tolerated and the
number of respiratory infections were significantly lower in vaccinated versus nonvaccinated SLE patients (0 versus 1 pneumonia, 1 versus 17 acute bronchitis, and 5
versus 21 viral respiratory infections) [179].
In 2008, however, Stratta et al. described one SLE patient with a se- vere flare-up
following flu vaccination, despite SLE were in a quiescent phase, not needing any
steroid or immunosuppressive therapy since 14 years [180].
In 2009, Holvast et al. analyzed the antibody response to influenza vaccination in 52
SLE patients and 28 healthy controls, by also studying the response to a booster
administered after four weeks [181]. In particular, seroconversion rate after the first
vaccination was 34.6% versus 28.6% to A/H1N1, 25% versus 10.7% to A/H3N2,
31
19.2% versus 10.7% to B strain in patients and controls, respectively. Seroprotection
rate and geometric mean titers (GMTs) increased similarly in patients and healthy
controls after vaccination, whereas the booster did not induce a rise of these values.
However, in patients not vaccinated in the previous year, GMTs and seroconversion
rate to A/H1N1 increased (from 34.6% to 46.2% with 5/52 [9.6%] negative SLE
patients at baseline who se- roconverted) after booster. Finally, the booster has been
demonstrated to be safe, considering that no SLEDAI modifications were noticed,
but useless, considering that no rise in seroprotection rate or GMTs was observed.
The same group in another paper analyzed the specific cell-mediated immune
response to A/H1N1 and A/H3N2 vaccine anti- gens through the identification of
IFN-γ secreting cells and reported a significant rise one month after vaccination in
54 SLE patients, even though at lower levels than normal controls [182]. The lower
cellular response was associated with the use of PDN and/or AZA.
In the same year, Wallin et al. [183] analyzed 47 SLE patients before and after flu
immunization. No SLEDAI modifications were observed and seropro- tection rate
was similar in patients and normal controls, whereas se- roconversion rate to A/New
Caledonia/20/99 strain was significantly lower in SLE patients than in normal
controls (57.4% versus 81.4%, p < 0.04); no significant difference, instead, was
observed for serocon- version rate to A/Wisconsin/67/2005 and
B/Malaysia/2506/2004 strains (61.7% and 51.1% versus 81.4% and 62.9%,
respectively). No inter- ference on antibody response has been observed for MTX
and AZA, whereas steroids were able to affect the response to one of the vaccine
antigens.
Finally, in 2010, Wiesik-Szewczyk et al. examined flu vaccine safety and
immunogenicity in 62 SLE patients and 47 healthy controls. In particular, one severe
and six mild/moderate exacerbations have been observed by three months from
vaccination. Moreover, 12 seronegative patients developed low-titer positivity for
anti-nuclear and anti-dsDNA antibodies one month after vaccination. Finally,
seroconversion rate for H1N1 A/New Caledonia/20/99, H3N2 A/Christchurch/28/03,
and B/Jangsu/10/03 strains one month after vaccination in patients versus healthy
32
controls was 53%, 55%, and 56% versus 83%, 85%, and 72%, respectively [184].
In conclusion, despite anecdotal cases of disease worsening after vaccination, inactivated flu vaccine, until now administered to 522 SLE patients (Table I), seems
generally safe, provided that quiescent patients are chosen. Treatment with PDN >10
mg/daily and AZA, but not MTX, however, may reduce antibody response to levels
that are considered protective.
33
2. Objective
To evaluate the safety and immunogenicity of the trivalent seasonal non-adjuvanted
anti-influenza vaccine in RA patients receiving different anti tumour necrosis factor
(TNF) agents or inhibitors of co-stimulus compared with patients with RA or SLE
receiving DMARDs and healthy controls and the effect of a simultaneous 23-valent
anti-pneumococcal or pandemic monovalent MF59-H1N1 adjuvanted vaccine.
Moreover, the effects of biological therapy on cell-mediated immunity following
influenza vaccination have been investigated.
3. Materials and Methods
Eighty-five RA patients, diagnosed on the basis of the American College of
Rheumatology criteria [185], age range 32–83 years, 68 females, under treatment
with TNFα blockers, Infliximab (14), Etanercept (33), Adalimumab (24) or the
inhibitor of co-stimulus Abatacept (14), have been vaccinated, after informed
consent, by intramuscular route with 0.5 trivalent non-adjuvanted split influenza
vaccine [Vaxigrip, Sanofi Pasteur MSD] for a total amount of 85 vaccine injections,
at least once during three consecutive influenza seasons (2008–2009, 2009–2010
and 2010–2011). In particular, 19, 30 and 36 patients were vaccinated in each
influenza season, 6 and 14 of whom represented a stable patient cohort vaccinated
respectively in the first and second, and in the second and third influenza seasons.
Flu vaccine contained 15 μg for each viral strain per dose: A/Brisbane/59/07 (H1),
A/Brisbane/10/07 (H3), B/Florida/4/06 in season 2008–2009, A/Brisbane/59/07
(H1), A/Brisbane/10/07 (H3), B/Brisbane/60/08 in season 2009–2010 and
A/California/7/2009 (H1), A/Perth/16/09 (H3), B/Brisbane/60/08 in season 2010–
2011.
Inclusion criteria were low-moderate activity (full joint count disease activity score
[DAS] < 3.7) [186] and stable (disease not needing any increase of therapy for at
least 6 months) disease, whereas exclusion criteria were any sort of contraindication
34
(referred allergy for egg or any vaccine component, pregnancy, etc.) at the time of
enrolment.
Forty-two age and sex-matched healthy controls (HC) received, after informed
consent, the same flu vaccine at least once during the above reported three
consecutive influenza seasons.
In the second season 30 RA patients (13 HC) received simultaneously a single dose
of
the
pandemic
monovalent
MF59-adiuvanted
influenza
vaccine
(A/California/7/2009 (H1)) [Focetria, Novartis].
In the first and third season, instead, respectively 19 and 15 RA (10 and 6 HC)
patients received simultaneously 23-valent polysaccaride pneumococcal vaccination
[Pneumovax 23, Merck].
In the last season 16 low activity (systemic lupus erythematosus disease activity
index [SLEDAI] <12 [174]) SLE and 9 RA patients under treatment with only
DMARDs (DAS < 3.7) were also enrolled.
All the patients were recruited among the outpatients attending the clinic of Clinical
Immunology and Rheumatology of the Sant'Andrea University Hospital in Rome.
All vaccinated RA patients were treated with low dose steroids (<10 mg/daily of
prednisone) and methotrexate (10-15 mg/weekly) or cyclosporine (<3 mg/kg/daily).
and presented stable disease activity [defined as the disease not needing any increase
of therapy for at least 3 weeks for SLE and 6 months for RA]. For SLE, all patients
that at the time of enrolment had disease activity involving central nervous system or
kidney have been excluded.
They underwent clinical and laboratory (specific anti-influenza virus antibodies,
antinuclear antibodies [ANA], peripheral blood lymphocyte [PBL] subpopulations)
evaluation before (T0) and 30 (T1) and 180 (T2) days after vaccination. Blood
samples were collected from HC at the same times.
35
3.1 Safety
Safety has been monitored at T0, T1 and T2 with:
1) DAS and SLEDAI for RA and SLE, respectively, to register possible
vaccine-induced disease reactivation.
2) All patients were given a diary card in order to register possible local and
systemic adverse reactions. Moreover, 1 week after vaccination all the
patients have been interviewed by telephone calls and asked about the
possible appearance of a list of clinical systemic and/or local side effects
including: shivering, fever (>37.5°C), headache, malaise, asthenia, arthralgia,
myalgia, local pain, redness, induration or swelling.
3) Laboratory evaluation, including erhytrocite sedimentation rate (ESR), C
reactive protein (CRP), blood cell count, Rheumatoid Factor (RF), Antinuclear antibodies (ANA)
The study was approved by local ethics committee.
3.2 Laboratory Evaluation
Specific anti-Influenza Antibodies
Sera were analyzed by hemagglutination-inhibition (HAI) test, according to standard
procedures. Antigens used for testing were the same or antigenically equivalent to
those included in the vaccine formulations. Each serum was tested at a starting
dilution of 1:10. Geometric mean titers (GMTs), seroprotection rate (SP) (the
percentage of vaccine recipients with a serum HAI titer of at least 1:40 after
vaccination), seroconversion rate (SC) (the percentage of vaccine recipients with an
increase in serum HAI titers of at least fourfold after vaccination) and
seroconversion factor (SCF) (the post-vaccination antibody titer divided by the pre-
36
vaccination antibody titer) were calculated. A seroprotection rate exceeding 70%
(60% in people aged >60 years), a seroconversion rate exceeding 40% (30% in
people aged >60 years) and a seroconversion factor exceeding 2.5 (2 in people aged
>60 years) were considered as cut-off levels of vaccine immunogenicity for adults
18–60 years old, according to the guidelines of the Committee for Human Medicinal
Products (CHMP). To fulfill the CHMP guidelines for the seasonal flu vaccination
each of the 3 vaccine antigens must meet at least one of the above criteria while all
criteria must be met for pandemic.vaccination [187].
Specific anti-Pneumococcal Antibodies
Their determination was performed only in the first season for antigens 4 and 14 one
month after pneumovax23 immunization. A positive immunization response was
defined as a 2-fold or higher increase compared with the prevaccination antibody
concentration. Since a protective level of serum antibody has not been strictly
defined and may differ among serotypes, these values were not used to identify
individuals with probable/possible protective antibody levels.
Levels of serotype-specific pneumococcal IgG to 4 and 14 were measured using
commercing available enzyme-linked immunosorbent assay (ELISA) for
quantitation of human IgG antibodies specific for S. pneumoniae capsular
polysaccharides. Briefly, ELISA plates were coated with Pn PS. Dilutions of human
sera absorbed with pneumococcal capsular polysaccharide were then added to the
ELISA plates. The serotype-specific antibodies were detected using goat anti-human
IgG antibodies conjugated with alkaline phosphatase, followed by addition of the
substrate, pnitrophenyl phosphate. The optical density was measured at 405 nm
using an ELISA plate reader. The optical density of the coloured end product is
proportional to the amount of anticapsular PS present in the serum. The lower limit
of detection was 0.01 mg/l.
37
Autoantibodies
RA sample sera were evaluated for ANA by indirect immune-fluorescence. (Alifax
Diagnostica S.p.A., Padova, Italy); rheumatoid factor (RF) was assessed by
nephelometry (Aeroset, Abbott Diagnostics, Abbott Park, IL, USA), according to the
manufacturer’s instructions.
Peripheral Blood Moninuclear Cells (PBMC)
Preparation of PBMC samples
PBMC were isolated from heparinized whole blood by density gradient
centrifugation using Ficoll-hypaque (Bio-Lynx, Brockville, Canada) and washed
three times in phosphate-buffered saline (PBS). Aliquots of 1×107 cells were
dispensed in Cryovials (Nunc) and placed in a freezing device (Nalgene).
Lymphocyte Subpopulations
Lymphocyte
subpopulations,
including
evaluation
of
regulatory
CD3+/CD4+/CD8−/CD25 bright/CD127 dimT cell frequency was performed on
patients and healthy controls vaccinated in the first flu season.
T-regs subpopulations were studied by a standard protocol based on five-color
immune-fluorescence flow cytometry analysis. Cells were surface stained using a
panel of fluorochrome-conjugated mAb including: PerCP-CD3, FITC-CD19, FITCCD4, APC-CD8, PE-CD45, PE-CD25, CD127 Alexa 647-conjugated (Becton
Dickinson, S.p.A.) and incubated for 30 min in the dark at room temperature.
Preparations were washed and fixed using the fluorescence-activated cell sorter
(FACS) lysing solution (Becton Dickinson Immunocytometry System, Mountain
View, CA, USA) according to the manufacturer's instructions. In particular, Tregs
38
have been identified by the following panel: CD3+, CD4+, CD8−, CD25bright,
CD127dim, and expressed as a percentage of CD4+ lymphocytes. Flow cytometry
was performed using a FACScanto 1 (Becton Dickinson Immunocytometry System,
Mountain View, CA, USA) flow cytometer. Forward and side light scatterswere
used to only include lymphocytes in the analysis. Twenty thousand lymphocytes
were tested for each sample. Data were analyzed by the dedicated “Diva 2.0
software”.
PMA-iono stimulated samples
PBMCs from 6 patients and 3 matched controls vaccinated in the second season at
T0, T1, T2 were simultaneously thawed and batch-processed. Cells were stimulated
with 20 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 μg/ml ionomycin
(Sigma-Aldrich, St. Louis, MO) at 37°C for 5 h to detect the frequencies of IL-17,
TNF-α and IFN-γ-producing T cells. Brefeldin A (1,5 μg/ml; Sigma) was added to
cultured PBMCs for 3 h. The stimulated PBMCs were washed in phosphate buffered
saline and incubated for 30 minutes at the dark and at 4 C° with CD4-Pe-Cy7, CD8APC-Cy7, CD69-PerCp.
Then PBMCs were fixed in 4% formaldehyde,
permeabilized with 0.1% saponin (Sigma), and stained with IFN--FITC, IL17a-Pe,
TNFα-APC. One hundred thousand events per sample were acquired using a FACS
Canto flow cytometer and analysis was performed with FACSDiva software (BD
Biosciences).
Non-antigen specific functional responses at three times points (IL-17a, TNFα and
IFNγ) were assessed gating on T lymphocytes, followed by gating on CD69+.
Among CD69+ we assessed the percentage of cells producing IFNγ, TNF- and IL17a.
39
4. Statistical Analysis
All statistical analyses were performed using the Stat Soft 6.0 and GraphPad
Software, Inc. program The SCF for the single subjects was derived in a logarithmic
(base 10) scale to obtain a roughly normal distribution and its statistical significance
between different groups was determined with a Student’s t test for unpaired data.
Statistical comparison of DAS, SLEDAI and mean percentages of lymphocyte
subpopulations was made by Mann–Whitney U and Wilcoxon tests for paired data.
Correlations have been explored by the Spearman's coefficient test. Ordinary and
repeated measures one-way ANOVA, with Bonferroni post-test correction have
been performed for variance evaluation of PMA-iono stimulated cells. P values
<0.05 were considered significant
40
5. Results
5.1 Safety
No significant increase of disease activity was found, in both SLE and RA groups,
before, 1 and 6 months after influenza vaccination.
Data shown for RA on biologicals are the mean of 3-year values, being
representative of the behavior observed in each year (Table 4).
No severe adverse reactions (life-threatening, requiring hospitalization and/or
intravenous treatment), were observed. Mild systemic and local side effects are
reported in table 5. No significant difference could be observed between vaccinated
patients and HC.
No statistically significant changes of the ANA titer, RF and inflammatory indices
have been registered.
Tab 4. DAS or SLEDAI for RA and SLE patients at T0, T1,T2 after flu
vaccination
RA BIO
RA
DMARDs
Patients N
85
9
Age mean ± SD (years)
55 ± 10
55 ± 10
Sex(F/M)
68/16
6/3
DAS or SLEDAI T0 mean ± 2.33
± 2.47 ± 0.2
SD
0.8
DAS or SLEDAI T1 mean± 2.28
± 2.52 ± 0.2
SD
0.8
DAS or SLEDAI T2 mean ± 2.24
± 2.3 ± 0.2
SD
0.9
SLE
16
38 ± 7
12/4
5.26
±
0.3
5.41
± NS
0.4
5.18
± NS
0.3
41
Tab 5. Systemic and local side effects in RA, SLE patients and HC
RA BIO
Redness/swelling injection
site
Fever (>37.5°C)
Headache
Asthenia/malaise
Arthralgia/myalgia
Chills
Total Events/N patients
5
4
6
7
12
3
37/85
(44%)
N patients with events/N 29/85
patients
(34%)
RA
DMARDs
0
SLE
HC
P
1
2
NS
0
1
2
2
0
5/9 (55%)
2
1
2
2
0
8/16
(50%)
4/16
(25%)
1
3
5
4
1
16/42
(38%)
10/42
(23%)
NS
NS
NS
NS
NS
NS
2/9 (22,2)
NS
42
5.2 Immunogenicity
Anti-influenza antibodies
Data on immunogenity observed in RA patients under biological agents during the
three seasons are shown in tables 6-8 while those observed in RA and SLE patients
on DMARDs, enrolled only in the last season, are shown in table 9.
2008/2009: RA patients did not fulfill any immunogenity criteria for
A/Brisbane/59/07 (H1), 1 for B/Florida/4/06, and 3 for A/Brisbane/10/07 (H3). HC,
instead, met all the 3 criteria for A/Brisbane/59/07 (H1) and A/Brisbane/10/07 (H3)
and only one for B/Florida/4/06. However, the GMT increase was not statistically
different between RA and HC for any antigen.
2009/2010: Both HC and RA patients fulfilled all the 3 immunogenity criteria for
the seasonal (A/Brisbane/59/07 (H1) A/Brisbane/10/07 (H3) B/Brisbane/60/08) and
the pandemic (A/California/7/2009 (H1)) antigens with no statistical difference
between them.
2010/2011: RA and SLE patients under treatment with DMARDs and HC fullfilled
all the 3 immunogenity criteria, while RA under treatment with biological agents
only 1 for A/Perth/16/09 (H3) and 2 for
A/California/7/2009 (H1) and
B/Brisbane/60/08.
In subjects immunized with only influenza increase in GMT versus H1(p = 0.008)
and H3 (p = 0.046) was statistically different between RA under biological therapy
and HC and versus H1 (p = 0.034) and B (p = 0.003) between RA under biological
therapy and RA under DMARDs.
Among HC and RA patients under treatment with DMARDs immunized also with
pneumococcal vaccine no response was observed vs H3 compared to the complete
response in subjects immunized with only influenza (for HC p = 0.034). Subgroups
analysis is shown in figure 1-2.
43
Tab 6. Immunogenity in season 2008-2009 for A/Brisbane/59/07 (H1),
A/Brisbane/10/07 (H3), B/Florida/4/06 in RA patients under treatment with
biologicals and HC
RA/HC
H3
H1
B
SP (%)
70/100
67/100
60/55
SC (%)
56/78
33/78
33/22
SCF
3/9
2,1/6,4
2,6/3
Tab 7. Immunogenity in season 2009-2010 for A/Brisbane/59/07 (H1),
A/Brisbane/10/07 (H3), B/Brisbane/60/08 and A/California/7/2009 (H1 pnd) in
RA patients under treatment with biologicals and HC
RA/HC
H3
H1
B
H1 pnd
SP (%)
88/83
80/100
100/91
84/83
SC (%)
72/81
68/45
68/54
81/54
SCF
4,6/6,9
5,6/3,5
5,8/4,4
12/7,3
44
Tab 8. Immunogenity in season 2010-2011 for A/California/7/2009 (H1),
A/Perth/16/09 (H3), B/Brisbane/60/08 in RA patients under treatment with
biologicals and HC
RA/HC
H3
H1
B
SP (%)
58/89
64/94
72/94
SC (%)
35/65
50/82
39/47
SCF
3/6
4/23
2,6/5,4
RA: rheumatoid arthritis; HC: healty controls; SP: seroprotection; SC: seroconversion; SCF:
seroconversion factor
45
Tab 9. Immunogenity in season 2010-2011 for A/California/7/2009 (H1),
A/Perth/16/09 (H3), B/Brisbane/60/08 n SLE and RA patients under treatment
with DMARDs and HC
SLE
SP
SC
SCF
H3
73
46
5,8
H1
87
86
31,5
B
87
46
6,3
RA DMARDs
SP
SC
SCF
78
45
7,2
89
89
26,6
78
78
20,6
HC
SP
SC
SCF
89
65
6
94
82
23
94
47
5,4
RA: rheumatoid arthritis; DMARDs: disease modifying anti-rheumatic drugs; HC: healty controls;
SLE: systemic lupus erithematosus; SP: seroprotection; SC: seroconversion; SCF: seroconversion
factor
46
Fig 1. GMT at T0 and T1 in subgroups immunized with influenza and
pneumococcal vaccines (P + I) or only influenza (I)
RA BIO I
RA BIO P+ I
100
100
80
80
60
60
40
40
20
20
T1
0
H1
T1
0
T0
H3
H3
B
T0
H1
HC P + I
B
HC I
500
500
400
400
300
300
200
200
100
100
T1
0
1
2
T1
0
T0
1
3
RA DMARDs P+ I
T0
2
3
RA DMARDs I
250
250
200
200
150
150
100
100
50
50
T1
0
1
T0
2
3
T1
0
1
T0
2
3
47
Fig 2. Seroconversion factor (GMT T1/GMT T0) in subgroups immunized with
influenza and pneumococcal vaccines (P + I) or only influenza (I)
6
5
4
3
2
1
RA BIO I
0
RA BIO P + I
H3
H1
B
16
14
12
10
8
6
4
2
HC I
0
HC P + I
H3
H1
B
40
35
30
25
20
15
10
5
RA DMARDs I
0
H3
RA DMARDs P + I
H1
B
No significant difference in vaccine response has been observed among the different
groups of biological agents (figure 3).
48
Fig 3. GMT at T0 and T1 in subgroups of RA patients under different
biologicals
ETANERCEPT
ADALIMUMAB
100
100
80
80
60
60
T0
40
T0
40
T1
20
T1
20
T1
0
T1
0
T0
H3
H1
H3
B
T0
H1
B
E
H3
H1
B
ADA
H3
H1
B
SP
70
70
80
SP
50
60
80
SC
22
44
22
SC
30
55
44
SCF
3
6,8
2
SCF
3,2
2,9
3,7
ABATACEPT
INFLIXIMAB
100
100
80
80
60
60
T0
40
T0
40
T1
T1
20
20
T1
0
H3
0
T0
H1
T1
H3
T0
H1
B
B
IFX
H3
H1
B
ABA
H3
H1
B
SP
57
57
42
SP
50
62
62
SC
57
57
42
SC
37
50
50
SCF
3,6
5,9
2
SCF
2,3
2,8
4
The cohort follow-up for repeated antigens revealed increase in GMT and
seroprotection rate for A but not for B antigens (Figure 4). In particular, in the 6 RA
49
patients vaccinated in the first and second seasons an increase in GMT at T0 and T1
has been observed for the reapeted antigens A/Brisbane/59/07 (H1),
A/Brisbane/10/07 (H3) (figure 4A) while in the 14 RA patients vaccinated in the
second and third seasons, only a slight increase at T0 and T1 has been observed for
A/California/7/2009 (H1 pnd) but a strong decrease in GMT for B/Brisbane/60/08
(figure 4b). When all the RA vaccinated population is taken in consideration the
seroprotection rate at T1 for the reapeted antigens reflects the above results (figure
4c).
50
Fig 4. A: GMT at T0-T1-T2 in the cohort vaccinated in the first and second
seasons (no 6). B: GMT at T0-T1-T2 in the cohort vaccinated in the second and
third seasons (no 14). C: Seroprotection rate in all the vaccinated RA patients
for the reapeted antigens.
2009
A
2010
2010
350
2011
B
350
300
300
250
250
200
H1
150
H3
H1N1
200
B
150
100
100
50
50
0
T0
T1
T2
T0
T1
0
T2
T0
T1
T2
T0
T1
T2
C
120
100
80
H1 A
H3
60
H1 B
B
40
20
0
2009
2010
2011
51
Anti-Pneumococcal Antibodies
Immunological response to pneumococcal vaccine (at least 2-fold or higher increase
compared with the prevaccination antibody concentration) has been observed in 7/19
patirnts (37 %) for antigen 4 and in 4/19 patients (21%) for antigen 14. In HC
immunological response has been observed in 4/9 (44%) for antigen 4 and in 2/9
(22%) for antigen 14.
5.3 Lymphocyte subpopulations
T-regulatory cells
In RA patients, Tregs were 2.7% of CD4+ lymphocytes (T0), 4% (T1) and 3.1 %
(T2). The increase from T0 to T1 was statistically significant (p=0.04), as well as the
decrease between T1 and T2 (p=0.04). In healthy controls, Tregs were 4.7% of
CD4+ lymphocytes (T0), 5.2% (T1) and 4.9% (T2). Differences among T0, T1 and
T2 were not statistically significant (Table 10). The same kinetics was observed for
the absolute values. The difference between RA patients and healthy controls in
Tregs (percent and absolute count) at T0 and T1 was statistically significant
(p=0.02; Table 10). In RA patients, the percent and absolute count of Tregs were
indirectly correlated with age (T0, r = 0.59; p=0.01) and directly with specific
antibody titer to influenza vaccine H1 (at T1, r = 0.6; p=0.004 and at T2, r = 0.6;
p=0.001) and H3 (at T1, r = 0.6; p=0.01 and at T2, r = 0.6; p=0.008). No significant
correlations have been observed between Tregs and DAS or ANA. A decrease in the
percent of CD19+, accompanied by a parallel increase of CD3+ and CD4+
lymphocytes, has also been found between T0 and T2 in both RA patients and
healthy controls
52
Tab 10. Vaccinated RA patients and healthy controls before (T0), 30 (T1) and
180 (T2) days after flu and pneumococcal vaccination in season 2008-2009
Lymphocite subpopulations
CD3+
CD4+
CD8+
CD19+
CD4+/CD27 high/CD127low
Vaccinated RA %
T0
69
41
28
11
2.7 b
T1
69
43
26
9
4a
Healty controls %
T2
68
44
24
12
3.1
T0
69
46
23
8
4.7
T1
69
45
24
11
5.2
T2
72
48
24
10
4.9
Data expressed as mean
a
T1vs T0, T1vsT2 p=0.04
b
T0 healthy controls vs T0 vaccinated RA patients, P=0.02
PMA-iono stimulated PBMC
A slight increase in activated cytokine-producing T cells at T1 compared to T0
followed by a reduction at T2 both in patients and controls have been found. Mean
values were higher in patients compared to controls at every time point (n.s.). In
particular, the dynamics of CD69+IFNgamma+ cell percentages was significant for
both patients and controls, whereas that of CD69+TNFalfa+ was significant only in
controls (Figure 5).
53
Fig 5. Percentages of activated cytokine secreting cells at T0-T1-T2 in RA
patients and HC
**
*
*
P < 0.05
54
6. Discussion
In this study, no significant differences in SLEDAI and DAS at 30 and 180 days
after flu non-adjuvanted seasonal vaccine, even administered simultaneously with an
adjuvanted pandemic or polysaccharidic pneumococcal vaccines, in the second and
third season respectively, have been observed. Such a relative safety of influenza
vaccine administration in patients with SLE and RA appears to be present even at
the immunological level, as inferred by the results of auto-antibodies measurement
and lymphocyte subpopulation analysis [188–189].
No severe side effects have been observed and the mild local and systemic adverse
events were not statistically different from HC, thus confirming the vaccine safety in
these patients [188–193].
The outstanding immune response observed in the second season also against the
non adjuvanted seasonal viral strains is brand new compared with our previous
experience on similar patient (20% identical) and control populations, in whom the
response resulted protective, but only partially meeting the CHMP criteria.
Probably the use of an innovative and effective adjuvant such as MF59, included in
the formulation of the flu pandemic vaccine, is the element able to make the
difference also towards the simultaneously, but not co-locally, administered non
adjuvanted flu seasonal vaccine, possibly for the innate immunity hyper-stimulation,
which helps the recruitment of T helper cells, broadly reacting with shared epitopes
among different viral haemagglutinins. MF59, on the other hand, may also be
responsible for the slight increase of local and systemic adverse events, mainly
among patients, probably because adverse events may mimic symptoms already
present in RA patients.
Adjuvanted vaccines, even though useful in immunosuppressed subjects, have
seldom been used in patients with autoimmune diseases (no more than 20%, [9]),
fearing disease reactivation.
Regarding specific immune response to pandemic influenza antigen, the current
study demonstrates that, despite patients and controls had no detectable antibody
55
levels before vaccination, thus confirming the low percentage of cross reaction
between pandemic and seasonal viral strains [189], specific post-vaccine antibody
values were achieved in far higher levels and patient percentage than previously
observed [138].
In other studies involving patients with rheumatic diseases only treated with
pandemic and not seasonal vaccine, antibody levels comparable to those obtained in
healthy controls have never been achieved after a single vaccine dose [190, 191,
194]. The observation of a specific antibody response to the pandemic one-dose
vaccine, which is higher than that observed in healthy controls for all the three
CHMP parameters, and the excellent response obtained towards the seasonal vaccine
strains too, suggest that these results are a consequence of a series of factors
including the MF59 adjuvant activity, the simultaneous pandemic and seasonal
vaccine administration, which may take advantage of the MF59-stimulated cross
reaction of apparently antigenically divergent haemagglutinins as well as of
previous natural and/or vaccine immunizations.
The reciprocal seasonal and pandemic cross reaction is also supported by data
obtained in normal adult and elderly subjects. Gasparini et al. [195], in fact,
observed a doubled antibody response to the seasonal H1N1 strain when pandemic
and seasonal vaccines were co-administered compared with the response obtained
with the seasonal vaccine alone. In this regard Tsai [196] reported that the MF59
adjuvant stimulates production of more broadly reactive antibodies against viral
strains that are mismatched to those in the vaccine.
Thus a cross reaction and protection even in high percentage of subjects is present
among apparently highly divergent viral strains, such as A[H1N1]pdm09 and
seasonal strains [194,197, 198], and these shared epitopes may be recruited and
stimulated with high efficiency by MF59.
To the best of our knowledge, studies analyzing the specific immune response to the
two simultaneously administered pandemic and seasonal vaccines in inflammatory
rheumatic diseases are still not present in the literature, whereas such studies have
been performed in type 1 diabetes and in healthy, including elderly, subjects [195,
56
199-200]. Zuccotti et al, in fact, in eighty type-1 diabetes young patients found an
overall 100% proportion of vaccinees with protective antibody titres one month after
vaccination, without significant difference between those who received either the
one- or the two-dose schedule.
The role of the adjuvant in stimulating specific immune response is also witnessed
by three studies reporting a statistically significant reduced GMT, seroconversion
and seroprotection rate in RA patients (partially anti-TNF treated) compared to
healthy subjects after non adjuvanted pandemic influenza vaccine administration
[146, 201, 202].
The aim of the adjuvant is to enhance the immune response in a population
expected to be mostly naïf to the antigens and to increase the amount of vaccine
produced by sparing antigens. Other potential benefits include better crossprotection against drifted influenza strains. MF-59 directly enhances antigen uptake
by activated dendritic cells, induces chemokine production and is involved in the
recruitment of cells to the tissues. In humans, the adjuvant-induced immune
response results in quantitatively higher antibody titres and high quality long-term
persisting antibodies. These results are probably related to the induction by MF-59
of strong CD4+ T-cell help and more germinal centre reactions [203].
Recently Khurana et al. demonstrated that MF-59 induces the epitope spreading
from HA2 to HA1, by which a much larger antigen number participates in the
immune response in contrast with the narrower recognition of the fragments of the
HA2 stem region by subjects vaccinated with the non-adjuvanted or with the alumadjuvanted vaccines, and increases the maturation of antibody affinity [204].
Seasonal and pandemic vaccines should be administered together or in close
proximity to obtain better response [205], whereas the seasonal vaccine
administration 3 weeks before the pandemic has been observed to be associated with
a lower response to the pandemic [206,207], data observed also by Gabay et al.
[194], independently of the time interval between seasonal and pandemic vaccine
administrations.
57
Response to seasonal influenza vaccination in RA and SLE patients under treatment
with DMARDs, vaccinated in the last season, is comparable to HC, according to the
literature.
The suboptimal response observed both in RA patients under treatment with
biological agents and HC in the first and third seasons could be partially due to the
interference of simultaneous pneumococcal vaccination.
Our preliminary in vitro studies, in fact, showed that the presence of the
polysaccharide vaccine inhibits specific and aspecific cellular immune response
towards proteic antigens in a dose-dependent manner. Larger studies to further
establish these findings are warranted.
The kinetics of a specific antibody response may probably be related to the Treg
dynamics after vaccination; a significant Treg increase has, in fact, been observed 30
days after vaccination compared to the baseline values, in direct significant
correlation with the specific anti-H1 and H3 antibodies, thus suggesting a fine
regulatory activity on the antibody response, probably consequent to the reequilibrium induced by TNFα blockers [208–208]. TNFα is able, in fact, to interact
with Tregs via some receptors expressed on their surface, improving their function
[209–211].
No studies have been published on Treg kinetics in immunized people to our
knowledge, except two papers on Bacillus Calmette-Guérin (BCG) [212] and
hepatitis B [213] vaccines, which report Treg increase. Data presented here seem
thus original and in line with the cited reports. The observed inverse correlation of
Tregs with age is in agreement with the literature [214] and confirms that the
regulatory lymphocyte subpopulation is an expression of such a delicate activity that
tends to progressively decrease with the growing age.
Cellular immune response plays a crucial role in clearing influenza infection,
furthermore in elderly people it correlates with protection more than serum antibody
response [215]. Recent studies reported normal cellular immune response in RA
patients treated with Rituximab [144] and in Wegener and scleroderma patients [216,
217], while it is diminished in SLE patients [182]. Cellular immune response to
58
influenza vaccine has never been investigated in RA patients treated with anti-TNF
or Abatacept. In the current study stimulated CD69 positive T cells presented a
similar profile between patients and healthy controls, with a cytokine pattern of T1
(IFN/TNF secreting cells) and T17 (IL17 secreting cells), and with a dynamics
showing an increase a month after vaccine stimulation and a decrease at lower than
baseline values at six months, which is coherent of what has been observed with T
regulatory cells
The increase in activated T cells producing IFNγ at T1 time point reflects the major
implication of TH1 response to vaccination. The non significant increase in T
activated TNF+ in patients is likely due to their theraphy. Il 17 does not seem
particularly involved in influenza response even though further studies are needed to
provide a definitive answer to this question.
59
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