Study design

The VaccHemInf project is a single-centre prospective, consecutive cohort of adult HSCT recipients transplanted at the Haematology department of a 5362-bed tertiary-care university hospital (Lyon, France). During the past 5 years, the 114-bed Haematology department carried out an average of 102 (range, 90–110) and 81 (range, 73-88) autologous and allogeneic HSCT yearly, respectively.

Setting

On referent haematologist’s agreement, eligible adult (autologous or allogeneic) HSCT recipients will be referred to the vaccination centre dedicated to immunocompromised patients hosted by the Infectious Diseases department of Lyon’s university hospital for inclusion in the VaccHemInf cohort. The recruitment period will be of 24 months and recipient overall follow-up will cover a period of 48 months. For the purpose of FIGHT, 30 control healthy volunteers will be enrolled and will be tested for immune functional assays. Healthy volunteers will be recruited among the donors to the blood bank of the Etablissement Français du Sang (EFS) of Lyon. According to the EFS standardised procedures for blood donation, informed consent will be obtained from healthy donors, and personal data will be anonymised at the time of blood donation prior to the transfer of blood to the research laboratory.

Participants

The workflow of the VaccHemInf cohort is described in figure 1. Recipients aged 18 years or more will be included. During the first visit (V1), explanations will be given on: (1) implementation of the vaccination schedule and preblood test assessment and (2) postvaccination assessments at the 3 months (V2), 12 months (V3) and 24 months (V4) visits to monitor vaccine response and tolerance. FIGHT immune functional assays will only be performed at V1 and V2, as described in the workflow presented in figure 2. Exclusion criteria will apply in case of post-transplant relapse of the haematological underlying disease or in case of recipients’ death.

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Figure 1

Workflow of vaccines and sampling schedule of the VaccHemInf cohort. FIGHT, Fonctionnalité Immunitaire après Greffe de cellules souches HématopoïéTiques; HSCT, haematopoietic stem cell transplantation.

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Figure 2

Workflow of the immune functional assays (‘FIGHT’) of the VaccHemInf cohort. EdU, 5-ethynyl-2’-deoxyuridine; FIGHT, Fonctionnalité Immunitaire après Greffe de cellules souches HématopoïéTiques; IFN-γ, interferon gamma; PBMCs, peripheral blood mononuclear cells; RT-qPCR, real-time reverse transcription PCR; TNF-α, tumour necrosis factor alpha.

Endpoints

To address the objectives mentioned above, the primary endpoint will be to measure vaccine-specific antibody titres and to express vaccine response in percentage of responders and geometric mean titres (GMTs) immediately before and at 3, 12 and 24 months after vaccinations.

Secondary endpoints will be: (1) to investigate immune status before and 3, 12 and 24 months after vaccinations through the routine immunomonitoring and FIGHT new immune functional assays; (2) to thoroughly document vaccine safety; (3) to implement an exhaustive prospectively maintained database including variables related to the above endpoints.

Variables

The following data will be exhaustively recorded in a VaccHemInf-specific numerical clinical record folder (e-CRF): demographics, comorbidities using the Haematopoietic Cell Transplantation Comorbidity Index15 and haematological underlying conditions (box 1). The transplant characteristics will include disease status at transplantation, prior transplantation(s), conditioning regimen, graft cellularity, donor type, donor HLA matching, stem cell source, graft-versus-host disease (GVHD) prophylaxis including post-transplant cyclophosphamide for haploidentical transplants, and donor and recipient serostatus for cytomegalovirus, Epstein-Barr virus and toxoplasmosis (box 2). The post-transplant characteristics will include time to engraftment, second graft when appropriate, occurrence and grade of acute GVHD and its treatment, occurrence and grade of chronic GVHD according to the last National Institutes of Health Consensus criteria and its treatment,16 17 opportunistic infections of bacterial, fungal, viral or parasitic origin occurring on conditioning (day 7 before HSCT) and after HSCT using the European Group for Blood and Marrow Transplantation classification for infections, infectious agent-related reactivations and diseases (updated with the latest guidelines, and which list and full definition criteria are provided in table 1),18–32 peripheral blood T-lymphocytes and B-lymphocytes subsets and natural killer counts (box 3). Event variables will correspond to the applied French vaccination schedule (table 2).7 The responses to vaccines are defined as follows: for pneumococcus, a 30–270 mg/L IgG titre corresponds to a healthy population range (supplier data). In immunocompetent adults vaccinated with the 23-valent non-conjugated pneumococcal vaccine, the median increase in IgG titre was 4.7 (IQR, 2.7–6.6) times.33–35 Consequently, a fourfold increase in the IgG titre will be considered significant to reach immunisation. For H. influenzae type b, serology has no definite critical value. In immunocompetent adults vaccinated with the H. influenzae type b conjugate vaccine, the median increase in IgG titre was 5 (IQR, 3.5–7) times.35 36 Consensually, values between 0.15 and 1 µg/mL will be considered to be short-term protective, with a linearity limit of 9 µg/mL.36 37 An IgG titre higher than the standard protection threshold will be considered protective for tetanus and diphtheria (≥0.1 IU/mL) and HBV (anti-HBs antibody titre >10 mIU/mL).38 An anti-HBs titre >100 IU/mL will be considered to confer long-lasting protection.39

FIGHT immune functional assays

To investigate innate and adaptive immune responses, two standardised immune functional assays will be carried out based on the findings of the REALISM project, which has established reference intervals in a large cohort of healthy volunteers.40 Briefly, the first immune functional assay will consist in collecting whole blood samples using the standardised TruCulture system (Myriad RBM, Austin, Texas, USA) directly containing immunogenic stimuli: medium alone (Null), lipopolysaccharide (LPS) at 100 ng/mL (from Escherichia coli O55:B5), and Staphylococcus aureus enterotoxin B (SEB) at 0.4 µg/mL, respectively.41 On a 24-hour stimulation at 37°C, the supernatant and the pellet will be collected using the separation valve. The supernatant will be stored at −80°C until batch serial quantification of cytokines tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) with Simple plex cartridges (ProteinSimple, San Jose, California, USA) using the ELLA platform (ProteinSimple). The cellular pellet will be stabilised in 2 mL of Trizol LS (Sigma Aldrich, Saint Louis, Missouri, USA) and the messenger RNA (mRNA) will be isolated with the NucleoSping 96 RNA tissue kit (Macherey-Nagel, Düren, Germany) for analysis of mRNA expression using the nCounter technology (NanoString Technologies, Seattle, Washington, USA), consisting in an RNA hybridisation method, whose gene set can include up to 780 genes.41 42

The second immune functional assay will consist in inducing T-lymphocyte proliferation and measuring IFN-γ release in the supernatant in response to an ex vivo stimulation with a standard mitogen, phytohaemagglutinin (PHA). Peripheral blood mononuclear cells (PBMCs) will be isolated from fresh, heparinized blood samples by Ficoll density gradient centrifugation (U-04; Eurobio, Les Ulis, France) and will be incubated with culture medium overnight. Then, PBMCs will be stimulated in duplicate with PHA at 4 µg/mL (R30852801; Remel, Oxoid, Thermo Fisher Scientific, USA) in a 96-well cell culture plate at 37°C for 72 hours. On incubation, supernatant will be harvested for IFN-γ quantification using the ELLA platform. Cells will be harvested and cell proliferation will be determined by the incorporation of 5-ethynyl-2’-deoxyuridine (EdU) at 10 µM for 2 hours in T-cells using the Click-It EdU AF488 flow kit (C10420; Life Technologies, Carlsbad, California, USA). In first, cells will be stained with allophycocyanin (APC)-labelled anti-CD3 monoclonal antibodies (Beckman Coulter, Brea, California, USA), fixed with IOTest 3 fixative solution (Beckman Coulter), and EdU will be activated with 100 µL of a reaction cocktail containing Alexa Fluor 488 (AF488) azide (component of the Click-It). Flow cytometry analyses will be performed on a Navios flow cytometer (Beckman Coulter). CD3⁺ cells will be first selected among total events based on a monoparametric CD3-APC histogram. Then, the percentage of EdU⁺ cells among CD3⁺ cells, along with the geometric means of fluorescence intensity of EdU-AF488 in this population, will be measured among 2.5×10³ CD3⁺ cells recorded.43

Using the immune functional assay described above, an innovative approach will consist in testing alternative recall antigens: varicella zoster virus (VZV) antigen (Microbix, Mississauga, Ontario, Canada), pneumococcal antigens (Pfizer, France) and tetanus toxoid (Pasteur Institute, Paris, France). The recall antigens stimulation will be performed on: (1) PBMCs, to quantify EdU+-cells and measure IFN-γ after 7 days of incubation; (2) whole blood samples for 24 hours in order to extract RNA (QIAamp RNA Blood Mini Kit, QIAGEN, France) and perform a reverse transcription using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific, France). TNF-α and IFN-γ gene expression will be quantified by qPCR using Taqman probes (Thermo Fisher Scientific) on CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, California, USA).

Routine cellular immunophenotyping will be processed at the Haematology and Immunology laboratories of Lyon university hospital (Lyon-Sud and Edouard Herriot hospitals, respectively). A large panel of B-cell and T-cell membrane markers associated with naïve or memory status will be investigated by flow cytometry. For B-cells, CD19+, CD20+, CD21+ and naïve B-cells (CD19+CD27-IgD+), memory B-cells commutated or not (CD19+CD27+IgD+/IgD-) will be counted. For T-cells, central memory T-cells (CD4+CD45 RA-CCR7+), effector memory T-cells (CD4+CD45 RA-CCR7- and CD4+CD45 RA+CCR7-) will be counted in addition of routine CD4+ and CD8+ subsets.

Sera from venous blood collection will be frozen at −20°C at the Institut des Agents Infectieux (Croix-Rousse Hospital, Lyon university hospital). Levels of specific serum IgG to seven serotypes included in the PCV13 vaccine (pn4, pn6B, pn9V, pn14, pn18C, pn19F, pn23F), and to pn1 and pn5 as markers of the immune response to PPV23, will be determined using a commercial ELISA (VaccZyme Anti-PCP IgG Enzyme Immunoassay Kit, Binding Site, UK). In addition, VZV-specific IgG and IgM (Laison VZV IgG and IgM, DiaSorin, Italy), diphtheria-specific and tetanus-specific IgG (VaccZyme Diphtheria or Tetanus Toxoid IgG Enzyme Immunoassay Kit) as well as HBV-specific anti-HBs antibody (anti-HBs2, Advia XP Centaur, SIEMENS, Germany) titres will be determined.

The biobanking of viable PBMC through cryopreservation will require a specific written consent. PBMC samples will be stored at the Centre de Ressources Biologiques Cytathèque (Lyon-Sud Hospital, Lyon university hospital). Furthermore, the FIGHT cohort will provide the opportunity to establish four different types of biobanks, enabling further explorations of innovative biomarkers: (1) TruCulture plasma and mRNA from cellular pellet biobank from whole blood stimulated with LPS, SEB or unstimulated, to study immune response; (2) EDTA plasma biobank to study viral reactivation markers and soluble host biomarkers; (3) RNA biobank to study new transcriptomic host biomarkers (RNA will be extracted from whole blood collected in PAXgene Blood RNA tubes (Thermo Fischer Scientific)); (4) viable freezed PBMCs.

Study sample size

The VaccHemInf project focuses on a highly specific population of patientsfor whom few or no vaccine efficacy data are available based on transplant characteristics. As the main goal of the study is primarily descriptive (prevalence of responders at different follow-up times), a sample sizing is not required. However, taking into account that prevalence is not known at the selected follow-up times and that logistic regression models will be used, we based our calculation on Machin’s rule of thumb suggesting the inclusion of 10 patients per estimated parameter in the model.44 For this reason, at least 50 patients are required.

Statistical methods

Qualitative variables will be expressed as count (percentage) while quantitative data will be expressed as median (IQR). The primary endpoint will be compared between V1 and V2, between V1 and V3 as well as between V1 and V4 regarding multiple variables related to haematological characteristics, treatment management, pattern of post-transplant immune recovery and transplant-related complications. Comparison will be performed using the Wilcoxon test, the Mann-Whitney U test, the χ² test or the Fisher exact test, where appropriate. For the GMT, means and 95% CI will be computed for the log10 transformed titres and then transformed back to the original units by exponentiation. For vaccine targets with low to moderate responder rates, the association between responders/non-responders endpoint and factor will be established using logistic regression. All factors associated with endpoint with a significance level p<0.15 will be kept for multivariate logistic regression models. These models will be adjusted with at most two or three confounding factors chosen according to evidence-based literature. In case of relapse of the haematological disease, in case of death during follow-up or in case of loss to follow-up, patients will be included in the analysis provided that there is at least the prevaccination and one or more postvaccination assessment available. Analyses will be based on two-sided p values, with statistical significance defined by p<0.05 and conducted with R V.3.4.4. Finally, appropriate multiple imputations (ie, at random or not) will be taken into consideration in the case of a relevant amount of missing values.

Patient and public involvement

No patients were involved in the design or implementation of this study. Study participants will be individually informed about their results during scheduled medical visits and will be given access, on demand, to the final publication of the study results.

Ethics and dissemination

The study is registered to the French Commission for Individual Data Protection and Public Liberties of Lyon’s university hospital under the number 18–063 and has been approved by the national review board for biomedical research in March 2018 (Comité de Protection des Personnes Sud-Est V) under the title VaccHemInf-69HCL17_0769.