Higher Memory Responses in HIV-Infected and Kidney Transplanted Patients than in Healthy Subjects following Priming with the Pandemic Vaccine

Background Memory responses require immune competence. We assessed the influence of priming with AS03-adjuvanted pandemic vaccine (Pandemrix®) on memory responses of HIV patients, kidney recipients (SOT) and healthy controls (HC). Method Participants (HIV: 197, SOT: 53; HC: 156) were enrolled in a prospective study and 390/406 (96%) completed it. All had been primed in 2009/2010 with 1 (HC) or 2 (patients) doses of Pandemrix®, and were boosted with the 2010/2011 seasonal influenza vaccine. Geometric mean titres and seroprotection rates were measured 12 months after priming and 4 weeks after boosting. Primary and memory responses were directly compared in 191 participants (HCW: 69, HIV: 71, SOT: 51) followed during 2 consecutive seasons. Results Most participants (HC: 77.8%, HIV: 77.6%, SOT: 66%) remained seroprotected at 12 months post-priming. Persisting A/09/H1N1 titers were high in HIV (100.2) and HC (120.1), but lower in SOT (61.4) patients. Memory responses reached higher titers in HIV (507.8) than in HC (253.5) and SOT (136.9) patients. Increasing age and lack of HAART reduced persisting and memory responses, mainly influenced by residual antibody titers. Comparing 2009/2010 and 2010/2011 titers in 191 participants followed for 2 seasons indicated lower post-2010/2011 titers in HC (240.2 vs 313.9), but higher titers in HIV (435.7 vs 338.0) and SOT (136 vs 90.3) patients. Conclusions Priming with 2 doses of Pandemrix® elicited persistent antibody responses and even stronger memory responses to non-adjuvanted seasonal vaccine in HIV patients than 1 dose in healthy subjects. Adjuvanted influenza vaccines may improve memory responses of immunocompromised patients. Trial Registration ClinicalTrials.gov NCT01022905

Duration of study (participants): from 1st vaccine dose to 4-6 weeks after the last vaccine dose.

Statistical Methods
Immunogenicity endpoints and confidence bounds will be calculated for each cohort, group and sub-group and compared (by Chi-square or Fisher exact test, and Wilcoxon's test or Kruskal-Wallis test) within a group, a cohort and across cohorts. The influence of variables will be tested in an univariate way.
A logistic model will be constructed to study the factors likely to explain the proportion reaching the primary endpoints. A multivariate analysis (logistic regression) will compare patients and healthy controls, while adjusting for other variables. The goodness-of-fit will be analysed using the Hosmer- Controls 1 X Patients 2 X 1 For control patients, to whom only 1 vaccine dose is now recommended, the 2 nd visit will take place 4-6 weeks after the first immunization. 2 To allow optimal comparisons with control patients, who will be recruited among the contacts of patients with cancer or rheumatologic diseases, we will ask patients from the cancer and rheumatologic disease cohorts whether they agree to provide an additional blood sample to measure there responses to the first vaccine dose. This additional venous bleed will be optional.

Justification for the proposed study a. Influenza A H1N1 disease
Influenza A H1N1 is a new virus that emerged in spring 2009 and rapidly spread around the world, causing a pandemic. Given the lack of herd immunity and the transmissibility of this new pandemic influenza A H1N1 2009 it is expected that a large proportion of the population, including those with underlying medical conditions will be exposed in the community. The evaluation of the morbidity / lethality caused by influenza A H1N1/09 is still ongoing. Nevertheless, it is recognized that the transmissibility and risk of this new influenza infection is high. The main complications of influenza are exacerbations of previous lung diseases or asthma as well as bacterial complications. Exacerbations of lung disease lead to frequent medical interventions, antibiotic overuse and hospitalizations. In addition influenza is known to cause viral pneumonia (a condition often unrecognized), which may be further complicated by bacterial pneumonia and/or Acute Respiratory Distress Syndrom (ARDS) and death. Other severe complications have been reported, among which myocarditis and central nervous events (seizures, encephalitis) are the most feared. In the US, UK, and Australia these complications required the hospitalization of up to 13% of cases with a positive A H1N1/09 test, lead to ICU admission in approximately 20% of them, and caused the death of 1-10 of 10'000 such patients.
These complications are fortunately rare in healthy individuals, but not so rare in patients with chronic diseases -as approximately 60% hospitalizations and most deaths occur in subjects vulnerable because of underlying risk factors.
The identified risk factors for influenza A H1N1/09 are similar to those that increase the risks of complications from seasonal influenza : young age (< 12 months, prematurity) and chronic diseases affecting the heart, the lungs or the immune system as well as pregnancy.
The WHO, the European Center for Disease Control and national health authorities world wide have thus recommended the priority immunization of high-risks patients. In Switzerland, the Commission fédérale pour les vaccinations recommends the priority immunization of pregnant women and of children from the age of 6 months, adolescents and adults with underlying diseases affecting their heart (cardiac malformation, pulmonary hypertension, cardiac insufficiency, etc.…), their lungs (asthma, cystic fibrosis, chronic pulmonary diseases, neurological or muscular disorders, etc.) or their Influenza vaccine reactogenicity and safety are essentially driven by the adjuvant -rather than by the specific antigens included in a vaccine. Thus, H1N1/09 vaccines will be licensed based also on the evidence collected 1) during the development of an H5N1 avian pandemic vaccine and 2) during the development and clinical use of the Novartis adjuvanted seasonal influenza vaccine (Fluad®). Fluad® has been distributed to more than 100'000 individuals (from newborns to seniors) during clinical studies and to 47 million individuals worldwide, which results into a large safety data base.
Importantly, these vaccines do not contain any live virus, such that they may be administered even to immunodeficient patients.
In contrast, these vaccines will be licensed with much more limited information regarding their immunogenicity / efficacy -as this is dependent upon the selected viral antigens. It is expected that 2 vaccine doses (at a minimal interval of 3 weeks) should result into a large proportion of healthy adults raising protective seroresponses -although the amplitude of this response and the exact proportion of those that will achieve protective titers is yet unknown. The Commission fédérale pour les vaccinations (CFV/EKIF) and the Federal Office of Public Health recommend that high-risk patients (with underlying cardiovascular or pulmonary disease or limited immune competence) be immunized as soon as the vaccines will have been licensed by Swissmedic (Bulletin OFSP, 20.10.2009).

c. Vaccine responses in patients at high risks of influenza complications
Protective vaccine responses will not be elicited by adjuvanted influenza vaccines in 100% healthy high risk group patients given the burden of prophylactic/therapeutic measures they require. Indeed, in the absence of the demonstration of positive vaccine responses, avoiding exposure as much as possible and empiric treatment with oseltamivir will remain required at each febrile influenza-like illness during many months -as influenza pandemic usually occur in several waves. Although correlates of protection have not yet been exactly defined for influenza A H1N1/09, antibody responses (assessed through inhibition of hemagglutination and/or microneutralisation) are known to largely mediate this protection and constitute the basis for the licensing of influenza vaccines.
In addition, this prospective collection of immune responses will provide original and most needed observations in the field. The ability of distinct patients populations with distinct degree and type of immunosuppressive conditions to raise vaccine responses to a specific common antigen have never been studied in such a systematic and standardized manner. The fact the targeted agent has not circulated in the population in the previous years is also an unique opportunity to assess essentially primary vaccine responses.

d. Safety of adjuvanted influenza A(H1N1) vaccines (admendment)
The safety of adjuvanted influenza A(H1N1) vaccines has been questioned by the media at large.
Fears that adjuvanted vaccines may have non-specific deleterious influences on the immune system and thus on underlying diseases have led some patients and physicians not to accept / recommend the use of adjuvanted vaccines to high-risk patients. After approximately 1'000 immunizations of study participants, our impression is that these vaccines are safe -which is also indirectly supported by the world-wide pharmacovigilance reports.
To more objectively demonstrate this vaccine safety in high-risk patients, we will include the evaluation of markers of underlying disease activity and/or graft function. Analyses will be performed at the final blood draw, and compared to baseline values retrieved from the medical chart (most recent control) or performed on serum harvested at Day 0.

Objectives: main questions to be answered through this research project
The general objective of this parallel-cohort study is to provide information on determinants that affect vaccine immune responses 1) within a given cohort (e.g. influence of CD4 T cell count among HIV-1 patients, of immunosuppressive regimen among patients with rheumatologic disorders, of time elapsed after liver transplant, etc).
2) as compared to healthy individuals 3) across the various cohorts -allowing to progress towards the identification of makers (or surrogates) of immune competence.
Towards this general aim, primary and secondary objectives have been identified.

Primary objectives : antibody responses
Which proportion of high-risks patients raise antibody responses associated with protection following one or two doses of influenza A H1N1/09 vaccines, as compared to healthy individuals after 1 dose ? What is the magnitude (titer) of their antibody response?
How is the proportion of seroresponders and the magnitude of the response influenced by age / underlying diseases / type of immunosuppression / number of vaccine doses? What is their influence on the magnitude (titer) of antibody responses?
Secondary objectives: T cell responses In which proportion of high-risks patients does influenza A H1N1/09 immunization induce a specific CD4 lymphocyte T cells response following 2 vaccine doses? How is this proportion and the magnitude of cellular responses affected by age / underlying diseases / type of immunosuppression? Does age / underlying diseases / immunosuppression affect the type of cytokines produced by vaccine-induced T cells?
What is the correlation between antibody and cellular responses? How does age / underlying diseases / type of immunosuppression affect this correlation.

Secondary objectives: vaccine safety
A vaccine safety objective has been added given the need to address vaccine fears by objectively demonstrating the safety of adjuvanted vaccines. This will be assessed by the proportion of patients with a deterioration of their medical condition, as reflected by increased requirements for immunosuppression, increased markers of underlying disease activity, or reduced graft function -as appropriate.
the first vaccine dose and 4 to 6 weeks after the last dose, to allow the assessment of vaccine responses.

Primary and secondary endpoints
Primary endpoints: Three co-primary immunogenicity endpoints after vaccination were chosen according to international guidelines used to evaluate influenza vaccines : 1) the proportion of subjects with serum hemaggglutination inhibition (HAI) antibody ≥ 1:40, 2) the proportion of subjects with either seroconversion or a significant increase in antibody titer, and 3) the factor increase in the geometric mean titer after 2 vaccine doses.
Secondary endpoints: The secondary immunogenicity endpoints will include 1) the same endpoints applied to the quantification of neutralizing antibody titers, 2) the proportion of subjects with detectable influenza A H1N1/09 specific CD4+ T cell responses after 2 vaccine doses, 3) the proportion of patients with a deterioration of their medical condition, as reflected by increased requirements for immunosuppression, increased markers of underlying disease activity, or reduced graft function -as appropriate.
Note : Assessing vaccine-associated adverse events in patients at high risks of influenza A H1N1/09 is not a primary objective of this study. However, according to good-clinical-practice guidelines we will monitor the frequency, duration and intensity of solicited adverse events during the 7 days after each vaccination and the incidence of serious adverse events until 4 weeks after the 2 nd vaccine dose. This will allow establishing potential correlations between inflammatory and antibody / cellular responses.

Randomisation and blinding
There will be no randomization or observer-blinding in this parallel-cohorts open-label study. However, all laboratory analyses will be performed by technical collaborators unaware of the patients' clinical conditions or treatment.

Study duration for subjects
Study duration for participants will be the time between the two study visits, i.e. between within 24h of the first immunization and the post-immunization visit scheduled 4-6 weeks after the last vaccine dose.
Immunization will include 2 doses at least 3 weeks apart -and 1 dose in healthy adult contacts of high-risk patients. Thus, study duration will be approximately 2 months and will not exceed 4 months.

Data collected in the CRF
Data collected in the CRF will include a list of demographic variables common to each cohort, as well as cohort-specific variables related to the underlying disease and treatment (cf Annex 3).
Information on the frequency, duration and intensity of solicited adverse events during the 7 days after each vaccine dose, and the incidence of serious adverse events until 4 weeks after the 2nd vaccine dose, will be collected as well (cf Annex 3).
Withdrawal procedure: Study participants will be free to withdraw from the study at any time, without having to provide any explanation. Routine care will not be affected by the study withdrawal, and pandemic H1N1/2009 will also be offered to patients not included into the present study.

Study population
This parallel-cohort study will include several cohorts -each led by a main investigator and his/her team of collaborators.
Remark: As vaccination is also recommended for pregnant women, women who correspond to the inclusion criteria of one of the cohort (HIV infection for example), will not be excluded from the study.
The vaccine used would then be Focetria® (as explained page 7).

HIV-infected patients (N max = 200)
Main investigator : Dr Alexandra Calmy  Complementary analyses: the number of viral copies/ml (HIV viremia) will be assessed at the final study visit (4-6 weeks after the last vaccine dose) and compared to baseline values extracted from the medical file (most recent control).

Patients with inflammatory rheumatic diseases (N max = 300)
Main investigator : Prof. Cem Gabay Patients will be recruited in the following groups (N = maximum number): Non-inclusion criteria : 1.
Refusal or failure to provide sufficient blood at least for the determination of antibody responses.

2.
Completion of the enrollment phase of the study (N max = 300).

Sample size
The influence of underlying disease and immunosuppression on vaccine-driven responses to influenza A H1N1/09 is unknown, precluding calculation of the sample size needed to ensure statistical power.
A maximal sample size of 300 subjects has been selected based on the number of patients actively followed. This number could be reduced by the interruption of enrolment, should subsequent available evidence indicate that a smaller sample size is sufficient. Note: all patients will receive 2 vaccine doses, allowing direct comparisons between patients of various treatment groups. We expect that a significant proportion of patients will consent to provide blood after their first vaccine dose, allowing direct comparisons with their healthy contacts.
Complementary analyses: C3, C4, thrombocytes, serum creatinin and anti-dsDNA antibodies (group 6, lupus), CRP, serum creatinin and ANCA antibodies (group 6, vasculitis) and CRP (groups 1-5) will be assessed at the final study visit (4-6 weeks after the last vaccine dose) and compared to baseline values extracted from the medical file (most recent control) or assessed on stored Day 0 samples. Patients with hematologic or solid malignancies may suffer from mild, significant or major limitations of their immune competence depending upon the disease condition and treatment regimen.

Patients with hematologic or solid malignancies (N max = 300)
Consequently, influenza immunization is recommended to all patients with cancer. In this study, we will offer a participation to patients suffering from disease-inducing immunosuppression (lymphoma), to those with severe chemotherapy-induced lymphopenia (glioma), to those with neutropenia-inducing Non-inclusion criteria : 1.
Refusal or failure to provide sufficient blood at least for the determination of antibody responses.

2.
Completion of the enrollment phase of the study (cf N max per group).

Sample size
The influence of underlying disease and immunosuppression on vaccine-driven responses to influenza A H1N1/09 is unknown, precluding calculation of the sample size needed to ensure statistical power.
A maximal sample size of 300 subjects has been arbitrarily selected based on similar studies and number of patients actively followed. This number could be reduced by the interruption of enrolment, should subsequent available evidence indicate that a smaller sample size is sufficient. Note: all patients will receive 2 vaccine doses, allowing direct comparisons between patients of various disease / treatment groups. We expect that a significant proportion of patients will consent to provide blood after their first vaccine dose, allowing direct comparisons with their healthy contacts. Patients who underwent solid organ transplantation may suffer from mild, significant or major limitations of their immune competence depending upon the time elapsed after transplantation and their immunosuppressive treatment regimen, which depends upon the type of transplant.

Solid organ transplant patients (N max = 250)
Consequently, influenza immunization is recommended to all patients with solid organ transplants.
Patients will be recruited in the following groups (N = maximum number): Non-inclusion criteria : 1.
Refusal or failure to provide sufficient blood at least for the determination of antibody responses.

2.
Transplant recipients after more than 5 years of transplantation, i.e. receiving minimal immunosuppression.

3.
Completion of the enrollment phase of the study (cf N max per group).

Sample size
The influence of underlying disease and immunosuppression on vaccine-driven responses to influenza A H1N1/09 is unknown, precluding calculation of the sample size needed to ensure statistical power.
A maximal sample size of 200 subjects has been selected based on the number of patients actively followed. This number could be reduced by the interruption of enrolment, should subsequent available evidence indicate that a smaller sample size is sufficient. Note: all patients will receive 2 vaccine doses, allowing direct comparisons between patients of various disease / treatment groups.
Complementary analyses: markers of liver function (ASAT, ALAT, bilirubin, gGT, alkaline phosphatase), of renal function (urea, creatinin), or of pancreatic function (C-peptide) will be assessed as appropriate at the final study visit (4-6 weeks after the last vaccine dose) and compared to baseline values extracted from the medical file (most recent control). Non-inclusion criteria : 1. Refusal or failure to provide sufficient blood at least for the determination of antibody responses.
2. Completion of the enrollment phase of the study. As influenza is moderately contagious, the risk of contagion 1) within a household, or 2) through health care professionals is markedly higher than through other social contacts. Consequently, household contacts and health care workers constitute a significant risk of transmission to high risk patients, such that their influenza immunization is recommended.
This cohort of healthy individuals will be recruited in the following groups: Non-inclusion criteria : 1.
Refusal or failure to provide sufficient blood at least for the determination of antibody responses.

2.
Completion of the enrollment phase of the study (cf N max).

3.
Acute or chronic disease likely to affect vaccine responses.

Study treatment
No drug nor vaccine will be administered for the purpose of this study.
Study participants will have been recommended an influenza A H1N1/09 immunization according to the official recommendations (recommendation for high risk patients: two vaccine doses (i.m.) at a minimal interval of 3 weeks). They will only be recruited after having decided to be immunized.
It is currently planned that Pandemrix® will be used for high-risk adult patients and their close contacts, and Focetria® for high-risk children. The vaccine name will be recorded in the CRF and treated as a variable. The batch number / expiry date will be recorded in the CRF ("page de garde").
Participation to the study will not change the patients' medical care / treatment.

Evaluation criteria
Central laboratory: All blood samples will be sent to a central laboratory (the laboratory of the Centre de Vaccinologie, CMU, Centre Médical Universitaire) for processing and serum/PBMC extraction. The samples will be identified by the patient number. Instructions necessary to ensure the appropriate storage and shipment of the sample will be provided on the laboratory form.
B cell immunity: This will be measured on cryopreserved sera, in the Laboratoire de Virologie (HUG).
Three co-primary immunogenicity endpoints after vaccination chosen according to international guidelines used to evaluate influenza vaccines are: 1) the proportion of subjects with antibody titers of 1:40 or more on hemagglutination-inhibition assay, 2) the proportion of subjects with either seroconversion or a significant increase in antibody titer, and 3) the factor increase in the geometric mean titer after 2 vaccine doses. These antibody responses will be assessed 1) within 24h of the 1st vaccine dose, for baseline antibodies (as H1N1/09 virus is already circulating in Switzerland, some patients may have been exposed prior to immunization), 2) within 4-6 weeks after the 2 nd vaccine dose.
Antibody responses to influenza are best measured by the quantification of functional antibodies. This will be assessed through hemagglutination inhibition (HI) and/or microneutralisation (MN) assays, :40 correlate best with clinical protection against influenza. It is expected that this will be similar following influenza A H1N1/09. This will be confirmed in the next few months.
Both assays may be generated with frozen serum, allowing the simultaneous evaluation of pre-and post-vaccination samples, thus reducing inter-assay variability. Taken into consideration the need for triplicates, a minimal serum volume of 200ul (0.5ml of blood) will be sufficient for the HI assay. A minimal serum volume of 1ml (2ml of blood) should allow performing both HI and MN assays. This will be specified within the next few weeks and necessary blood volumes reduced to as low values as possible.
T cell immunity: This will be measured on cryopreserved PBMCs, in the laboratories of the Centre de Vaccinologie, CMU. The secondary immunogenicity endpoint was selected as the proportion of subjects with detectable influenza A H1N1/09 specific CD4+ T cell responses after 2 vaccine doses.
Indeed, cellular responses may be affected differently than antibody responses by age, underlying diseases and immunosuppression.
To assess vaccine-induced T cell immunity, we will first try to adapt to influenza A H1N1/09 a wholeblood assay measuring the frequency of influenza-specific CD4+ T lymphocytes. Briefly, whole-blood samples will be stimulated in vitro during 6h with influenza A H1N1/09 (or control influenza viruses) in the presence of anti-CD28 and anti CD49d co-stimulating antibodies. Brefeldin A will be added to block extracellular cytokine transport during the last 4h of incubation. Following lysis of red blood cells, lymphocytes will be permeabilised and stained using fluorochrome-conjugated monoclonal antibodies against CD4, CD69 (as activation marker) and IFN-γ or IL-2 prior to assessment of the frequency of influenza-specific activated CD4 T cells by multiparameter flow cytometry. Frequencies of responding cells will be defined as percentages of the total positive events, and considered as reflecting frequencies of influenza-specific CD4 T cells. We will attempt the optimization of this assay, based on our expertise with the detection of varicella-specific vaccine responses, with as little as 3ml of whole blood. Vaccine-induced influenza-specific CD4+ T lymphocytes may however be too few to be identified through a whole blood assay. Should this be the case, we would work with purified peripheral blood mononuclear cells. Briefly, mononuclear cells would be isolated, centrifuged and frozen. After thawing, cells would be restimulated in vitro with influenza A H1N1/09 (or control influenza viruses) prior to fixation, staining and analysis of cytokine producing cells (IFN-g, IL-2, etc.) by flow cytometry. This assay is much more sensitive but requires a higher volume of blood (minimal amount to be defined). Results will be expressed as numbers of specific cytokine-producing-cells. Direct correlation between cellular responses and protection are not yet established, although these cellular responses are considered as conferring some degree of protection even in the absence of neutralizing antibodies.
The minimal volume of blood required for the analysis of cellular responses will be defined during the next few weeks. Blood sampling will be limited by the age of the patient (total of 5ml in young children, 10 ml in older children, 30ml in adults). Thus, T cell responses will only be determined in patients for whom enough blood will have been harvested.
Vaccine-associated adverse events: Studying vaccine reactogenicity / safety is not an objective of this study. However, any vaccine study is expected to offer the follow-up of potential adverse events to study participants. Parents or patients will be invited to record on a diary card temperature, potential local or systemic reactions to each vaccine dose as well as any unexpected event. They will be offered medical advice if in doubt concerning the occurrence of a potential vaccine-associated adverse event. It is expected that immunosuppressed patients will raise weaker inflammatory responses than healthy adults. As inflammatory reactions reflect innate responses and shape adaptative, B and T cell immunity, we will seek for potential correlations between inflammatory and immune responses.
All solicited local adverse events will be considered as related to the H1N1 vaccine, whereas the clinical investigators will assess the causality of solicited or unsolicited systemic adverse events.
Underlying disease and treatment: Information on the underlying disease of these high risk patients will be collected through a medical history and by assessing their medical file. Data will include demographic, clinical characteristics, with a special focus on the degree of immunocompetence, and treatment at time of immunization.
To more objectively assess any potential changes in underlying disease, blood markers of underlying disease activity / graft function will be assessed at the final study visit and compared to baseline values.

Data collection and management
Source of data: Information relative to the patient underlying disease and/or treatment will be retrieved through a medical history taken at time of enrollment and completed by access to the patient medical record -as needed. Paper-based CRF will be considered as source data.
CRF: A paper-based CRF will be used to facilitate enrollment and data collection at several locations within the HUG. Subjects will be identified on study documents by the first two letters of their first and last names and by a study number. Subjects will not be identified by name on any study document. This CRF will include common items to all participants as well as cohort-specific items. For each specific cohort, only the common and cohort-specific items will be included in the printed paper-based CRF, ensuring that inappropriate items are not included. All CRF data will be entered legibly in black ink. Amendments and errors on the CRF will not be erased, covered with correction fluid or completely crossed-out; rather a single line will be drawn through the error and the correction will be initialled and dated by the investigator or authorized personnel. CRFs will be currently maintained, reviewed, clarified and processed in accordance with the Standard Operating Procedures and working practices.
The CRF has now been generated by clinical research professionals (Electricpaper S.A., http://www.electricpaper.de/produkte/teleform.html) so as to allow processing by the Cardiff Teleform Intelligent Document Capture and Processing system (http://www.cardiff.com/products/teleform/), ensuring their adequate transfer into a single common ACCESS data base. The database has also been constructed by Electricpaper S.A., ensuring its adequacy with the CRFs.
Documents storage and keeping: All documents, including the original study protocol, notification letters from the IRB and Swissmedic, paper-based CRFs will be kept secure in accordance with the good clinical practice guidelines and applicable regulatory requirements.

Adverse events management
No drug nor vaccine will be administered for the purpose of this study.
However, study participants will have been recommended by their physician an influenza A H1N1/09 immunization with a licensed influenza A H1N1/09 vaccine. Any potential serious vaccine-associated adverse event will be notified by the physician in charge of the patient, according to Swissmedic's instructions. Should the study investigators be made aware of a missing notification, they would report it immediately and follow Swissmedic instructions.
To note: the immunization of high-risk patients will be taking place throughout Switzerland (and many other countries) at the same time. Vaccine-safety monitoring will thus be ongoing at a national and international level, providing information on potential vaccine-safety signals.

DEFINITION
An adverse event (AE) is defined as any adverse change from the patient's baseline condition. An adverse event does not necessarily have a causal relationship with the study treatment. A detailed description of any solicited adverse event will be entered in the CRF, including its intensity graded on a 6-point scale (mild (1-2), moderate (3-4), or severe (5-6)), the action undertaken for this event, the investigator's assessment of the relationship of the event to treatment, and the outcome. All serious Influence of age, underlying diseases and immunosuppression on vaccine responses to influenza A H1N1/09 immunization Version n°4, 20.07.2010 -Amendment 3 page 23/33 adverse events encountered during the clinical trial will be followed up until the event is either resolved or adequately explained.
A serious adverse event (SAE) as defined by ICH is any untoward medical occurrence that at any dose meets any of the following conditions: -Results in death -Is life-threatening (the subject was at risk of death at the time of the event. It does not refer to an event which hypothetically might have caused death if it were more severe) -Requires or prolongs hospital stay significantly -Results in persistent or significant disability/incapacity, -Constitutes a congenital anomaly/birth defect -Is an important medical event Important medical events are those that may not be immediately life threatening, but are clearly of major clinical relevance. They may jeopardize the subject, and may require an intervention to prevent one of the other serious outcomes noted above.
The nature of the SAE (expected or unexpected) as well as the relationship between the SAE and the study drug (imputability) will we assessed in order to define whether it is a Suspected Unexpected Serious Adverse Reaction (SUSAR) or not. Reports of all SAE will be submitted to the Ethics Committee and SwissMedic within 15 days for the SUSAR (as soon as possible for a death and within 7 days). The non-SUSAR SAE will be reported by the way of annual reports.

Quality control and assurance
This study will be performed according to GCP guidelines through a joint effort of senior investigators with extensive experience working with high risks patients, carrying out clinical investigations, working with influenza viruses and/or assessing complex vaccine responses.
Patients will be recruited at time of a routine medical visit in the respective Divisions of the Geneva University Hospital, by their physicians. The physicians will be instructed to provide adequate information and explanation regarding the aim, procedure and potential benefits and side effects of the study to their patients.
Professional expertise has been secured to prepare the CRF and the related data base. A data manager will be responsible for electronic data transfer and handling of the data base. Blood samples An expert biostatistician (Dr Combescure) will be responsible for all statistical analyses. All investigators will contribute to the analysis and interpretation of the data, and to their publications.

Reporting Procedures
• Serious adverse events, see above • Amendments to the protocol. Minor amendments (typographical errors, changes in administrative procedures and the like) will be bundled and submitted to the Ethics Committee at regular intervals of three months. Substantive changes will be formulated as study amendments and submitted to the Ethics Committee, and to Swissmedic, following the legal procedures as outlined on the Swissmedic website.
• Reports. The study duration for the participants is 12 weeks, but data generation and interpretation will be long. We expect the study to be terminated within 2 years. If this is not the case, and intermediate report will be produced and transmitted to the Ethics Committee and Swissmedic one year after the first patient starts the study. If the expected delays are kept, a Final Report will be produced two years after the study start.

Clinical Site Monitoring/Audit
Personal medical data may be reviewed at clinical sites by properly authorised individuals as part of monitoring or audit of the trial, but such information will be treated as strictly confidential and will in no circumstances be made publicly available.

Number of subjects
A maximum of 1500 subjects (including 250 controls) will be enrolled in 6 parallel cohorts including patients with HIV-1 infection (n=200), rheumatologic disorders (n=300), malignancies (n=300), solid organ transplant recipients (n=250), high-risk children (n=200) and healthy contacts (n=250). We expect a difference in the percentage of responders between the control group and the other groups, but the magnitude of this difference is presently unknown. Given the sample size, we assessed the statistical power of the comparison of the response rates, assuming a response rate of 83% in the control group [Clark W, 2009], and 68%, 73%, and 78% in the groups of patients (corresponding to differences of 15%, 10% and 5% respectively). The risk α was 5% (two-sided).
The results are presented in the following Overall, the planned sample size will provide a statistical power ranging from 70% to 79% for an expected difference of 10% in the response rate. The power is systematically beyond 95% for an expected difference of 15%. Given that many patients are markedly immunosuppressed, we do expect that differences will indeed be higher than 15%.
Assuming a response rate of 91% (after 2 doses of unadjuvanted vaccine), and differences of 15%, 10% and 5%, the power will be that described in the following The planned sample size will provide a statistical power ranging from 85% to 91% for an expected difference of 10% in the response rate. Given that many patients are markedly immunosuppressed, we do expect that differences will be higher than 10%.

Data analysis
The general objective of this parallel-cohort study is to provide information on determinants that affect vaccine responses: -in each cohort (i.e. HIV, rheumatology, oncology, transplant, pediatric, healthy controls), e.g.
influence of CD4 T cell count among HIV-1 patients, of immunosuppressive regimen among patients with rheumatologic disorders, of time elapsed after liver transplant, etc.
-as compared to healthy individuals -across the various cohorts -allowing to progress towards the identification of makers of immune competence. Proportions and titers will be compared (by Chi-square or Fisher exact test, and Wilcoxon's test or Kruskal-Wallis test) between the groups and the controls.
The influence of potentially relevant variables will be tested in an univariate way. A logistic model will then be constructed to study the factors likely to explain the proportion reaching the primary endpoints: this will include the subgroups plus the variables with an effect in the previous analysis. An interaction between the subgroups and other factors will be considered if necessary. A multivariate analysis (logistic regression) will be performed to compare high-risk patients and healthy controls, while adjusting for other variables. The goodness-of-fit will be analysed using the Hosmer-Lemeshow' test. will be also performed. For this purpose, we will transform the CD4 T to get a gaussian distribution.
The quality of model will be analysed (R-square, interpretation of residuals).
Recorded solicited local and systemic adverse events during 7 days after each vaccination will be described and compared using non-parametric methods. Similarly, changes in markers of underlying disease activity / graft function will be described and compared across study groups.
All the tests will be two-sided with a significance level at 5%.
Addendum: given the good immunogenicity of adjuvanted vaccines, Swissmedic has defined that a single vaccine dose will be sufficient for healthy controls. Thus, we will assess which are the determinants that allow 2 vaccines doses in immunosuppressed patients to raise responses similar to one dose in healthy adults. Comparison of responses to the first vaccine dose will be assessed in the rheumatology and oncology cohorts, where patients will be asked to provide an extra sample.
Although this will be optional, we expect most patients to accept this procedure, a routine in their treatment.

Dropouts and missing data management
The per-protocol immunogenicity analyses will only include subjects with available blood samples before and after immunization -and include a complete set of analyses.
The intention-to-treat analyses will include all subjects who provided a post-immunization blood sample -even in the absence of a pre-immunization sample. The pre-post immunization comparisons will not be possible and the interpretation of vaccine responses will have to take into consideration the likelihood of pre-immunization exposure to influenza A H1N1/09.
Subjects dropping out of the study prior to the post-immunization blood sampling will not be included in the statistical analyses. Patients will be replaced if there is no vaccination.
Reasonable attempts will be made to limit the amount of missing data related to serious adverse event and patient outcomes to ensure that important information related to the primary objective of the study is captured. In case of missing data, we will first try to retrieve the information in the patient databases of the University Hospital of Geneva. No imputation will be made on the CRF for missing data and further details of how missing data has been handled will be outlined in the study analysis plan.
Variables with less than 20% of missing data will be kept in the analyses, and the patients with missing data will be excluded from the multivariate analyses. Variables containing more than 20% of missing data will not be included in the multivariate analyses and a logistic regression model will be performed to identify the factors associated with missing data.

Criteria for the termination of the trial
The trial will be terminated whenever the maximal sample size has been reached or whenever the available evidence indicates that increasing the sample size would not improve statistical significance any further.

Risks and benefits
The only risks associated to this study are those of venous puncture (hematoma, etc.).
The potential benefits include the identification of patients who succeeded or failed to generate vaccine responses to influenza A H1N1/09 vaccines. This information will provide relief to responding high-risk patients (or their parents) and identify non-responding patients for whom strict prophylactic / therapeutic measures would remain required despite immunization. Thus, this study confers direct benefits to the participants.
In addition, it will provide most needed information on the protection that adjuvanted influenza vaccines may offer to high-risk patients with underlying diseases and various levels of immune competence. This is an important indirect benefit for all high-risks patients.

Ethical approval
This study was submitted and approved by the relevant institution Ethical Committee (Comité d'Ethique de Médecine interne, Comité d'Ethique Mère-Enfant).
It will not start before official approval by Swissmedic.

Information and consent
Written consent will be obtained from all patients (or from parents or legal guardians, and children whenever possible) before any study procedure (i.e before the first blood sample).

Confidentiality
All records will be stored in secure files and computers. Data will be anonymized for analysis and publication. Only the investigators and legal authorities will have access to the data.

Publication policy
This study will be registered in the NIH international registry of clinical trials. It is intended for (several) publications in scientific journals with peer-reviewed editorial policy. Study results will be shared as confidential privileged information with Swissmedic and the Federal Office of Public health at time of manuscript approval.
Individual patient results will be communicated to the physician in charge.

Financing and insurance
This study will be run independently from the pharmaceutical industry, so as to increase its perceived credibility.
Patient enrolment, data collection of underlying disease / treatment and pre-immunization venous sampling will be supported by existing resources from the investigators of each specific cohort. The costs of data acquisition (CRF), data management and data analyses as well as vaccine response analyses will be supported by existing research funds of the Center for Vaccinology and Neonatal Immunology (Prof. Claire-Anne Siegrist) and by an advanced researcher research grant submitted to the Centre de Recherche Clinique. Additional research funds will be seeked by the investigators to support the determination of T cell responses in as many participants as possible.
There will be no financial incentive to patients or participating physicians, to whom the (expensive) determination of vaccine responses will be offered free of charge. To note, the costs of influenza A H1N1 immunization will be free in Switzerland for all high-risk patients and their contacts, with no franchise nor participation (quote-part).
The study participants will be covered against the risks associated to this study by an insurance issued to the Hôpitaux Universitaires de Genève.

2010 study extension: immune memory
Although analyses are ongoing, the follow-up of 766 immunocompromised adult patients and 133 controls demonstrated that : -adjuvanted vaccines were well tolerated, inflammatory reactions being fewer / less intense in immunocompromised patients than in healthy controls and severe adverse events being essentially non-associated to immunization (see interim report in Annex); immunization did not trigger autoimmune disease exacerbations nor graft rejection and did not require the intensification of immunosuppressive treatments. A transient increase of HIV-1 viremia was noted, as previously described following seasonal influenza immunization, but remained without clinical influence.
-adjuvanted vaccines elicited potent antibody responses after a single vaccine dose in healthy individuals; similar responses were observed after 2 doses in HIV-infected patients; however, vaccine responses remained significantly lower in transplant patients.
In October 2010, seasonal influenza immunization will be recommended to these high-risk patients.
They will receive a non-adjuvanted vaccine including 3 influenza strains:

Note : HIV-infected patients with increased viremia would be invited to return for a medical visit 3 months after immunization to confirm that viral control is appropriate .
Antibody responses (and HIV-RNA viral load) will be measured and compared as previously described.
We will use the same CRF / carnet des vaccinations to obtain an up-to-date medical history and follow-up potential vaccine adverse events.

Inclusion criteria :
1) Adult patients with HIV-infection, after solid organ transplant or healthy (for example health care professionals) to whom 2010 seasonal influenza immunization is recommended 2) Who were immunized against influenza A/H1N1 in the fall of 2009 3) Who are willing to return for a second visit 30 days later 4) Who have signed an informed consent.

Exclusion criteria :
1) Refusal or failure to provide blood (minimum volume 2 ml).

Risks and benefits
Risks : Those of venous bleeding: bruising at the site of injection may not be excluded.
Indirect benefits: Improvement of the knowledge, allowing defining whether / which immunocompromised patients should receive adjuvanted seasonal influenza vaccines in the future.

Statistical evaluation
This will be a descriptive analysis of the influence of age and immune suppression on the induction of immune memory by adjuvanted versus non-adjuvanted influenza vaccines. This influence is yet unknown, precluding sample size estimation. However, data collected in 2009 confirmed that the inclusion of approximately 150 patients by group was sufficient to identify significant differences between healthy and immunosuppressed patients.
Analyses will be performed as previously described and found appropriate.

Funding
The costs of this study extension will be covered by existing research funds and new sources of funding (pending demand to the Swiss HIV Cohort Study).

Ethical approval
This extension should ideally start on October 1st (influenza immunization campaign), but will not be initiated prior to its approval by the Ethical Committee and Swissmedic.