Maria Mamani-Matsuda

Antonio Cosma

Sandra Weller

Ahmad Faili

Caroline Staib

Loïc Garçon

Olivier Hermine

Odile Beyne-Rauzy

Claire Fieschi

Jacques-Olivier Pers

Jacques- Olivier Pers

Nina Arakelyan

Bruno Varet

Alain Sauvanet

Anne Berger

François Paye

Jean-Marie Andrieu

Marc Michel

Bertrand Godeau

Pierre Buffet

Claude- Agnès Reynaud
email: reynaud@necker.fr

Jean-Claude Weill
email: weill@necker.fr

The human spleen is a major reservoir for long

Keywords: thrombocytopenic purpura, MVA, modified vaccinia Ankara virus, RIT, rituximab treatment

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Long-lived memory B cells and specific antibodies have been reported to persist in the body for decades after specific vaccination. [START_REF] Crotty | Cutting edge: long-term B cell memory in humans after smallpox vaccination[END_REF][START_REF] Hammarlund | Duration of antiviral immunity after smallpox vaccination[END_REF][START_REF] Amanna | Duration of humoral immunity to common viral and vaccine antigens[END_REF] One of the central questions concerning this phenomenon concerns the maintenance of this memory in the absence of repeated stimulations with the original vaccine. Experiments in mice suggested that the survival of memory B cells depended neither on constant cell proliferation nor on the presence of the immunizing antigen. [START_REF] Maruyama | Memory B-cell persistence is independent of persisting immunizing antigen[END_REF][START_REF] Schittek | Maintenance of B-cell memory by long-lived cells generated from proliferating precursors[END_REF] It was therefore assumed that these cells remain quiescent for long periods of time, but nonetheless react very rapidly if they encounter the immunizing antigen. It has also been suggested that the presence of specific serum antibodies is directly linked to the presence of the corresponding memory B cells. In this model, each new antigenic stimulation provides bystander T cell help for the previously generated memory B cells, leading to the differentiation of some of these cells into Ig-secreting plasmocytes. [START_REF] Bernasconi | Maintenance of serological memory by polyclonal activation of human memory B cells[END_REF] An alternative explanation for the continued presence of serum antibodies a long time after the initial pathogen encounter has also been put forward, according to which each vaccination induces both the immediate differentiation of plasmocytes and the formation of long-lived plasma cells. [START_REF] Manz | Lifetime of plasma cells in the bone marrow[END_REF][START_REF] Slifka | Humoral immunity due to long-lived plasma cells[END_REF] These memory plasma cells would survive in bone marrow and would be replaced, at a certain rate, by plasmablasts generated in response to new pathogens. [START_REF] Odendahl | Generation of migratory antigen-specific plasma blasts and mobilization of resident plasma cells in a secondary immune response[END_REF] Smallpox vaccination is considered an ideal model for studies of the basis of long-term cellular and humoral memory. [START_REF] Slifka | Immunological memory to viral infection[END_REF] Vaccinia virus was successfully used to eradicate variola virus, the etiologic agent of smallpox. As smallpox vaccination was stopped in the mid 1970s and smallpox was eradicated worldwide in 1977, [START_REF] Smith | Smallpox: anything to declare?[END_REF] we can assume that people have not been re-exposed to the pathogen since then. Using ELISPOT assays following in vitro cell stimulation, we studied the maintenance of the vaccinia virus-specific memory B cell pool in the blood and spleen of normal individuals, and in those of patients who had undergone B cell-targeted immune interventions. We show here that long-lived memory B cells have their own central reservoir, the spleen, and seem to display their own homeostatic regulation, independent of the presence of the corresponding plasmocytes.

Materials and Methods

Viruses

The modified vaccinia virus Ankara (MVA), which was used as the antigen in the B-cell ELISPOT assay, was amplified in chicken embryo fibroblasts and purified by centrifugation on a sucrose cushion. Alternatively, highly purified MVA preparations for use in ELISA were produced by centrifugation on two successive sucrose gradient cushions. Titers were determined by plaque assays on confluent chicken embryo fibroblast monolayers grown in six-well tissue culture plates. [START_REF] Staib | Transient host range selection for genetic engineering of modified vaccinia virus Ankara[END_REF]

Patients and controls

Approval for this research was granted by the "Comité de protection des personnes Ile-de-France II". Written informed consent was obtained from all patients. The characteristics of the subjects, including age, sex, diagnosis and medication taken at the time of sample collection are detailed in Supplemental Tables S1-S3. Nine splenectomized patients (V10 to V18: 4 benign and 3 malignant pancreatic tumors, 2 idiopathic thrombocytopenic purpura, ITP) displayed normal numbers of B cells and had not undergone any chemotherapy. Nine rituximab-treated patients (RTX1 to RTX9) suffered chronic ITP, Sjogren's syndrome or non-Hodgkin's lymphoma. Rituximab regimen is detailed in Supplemental Table S2 (3 patients received light corticotherapy). Chemotherapy treatment, when given (RTX3, RTX4), was stopped at least 25 months before sample collection, except for V19. Bone marrow samples were obtained from diagnosis aspirations. For all patients, B cell content and proportions of IgG + B cells are detailed in Supplemental Tables S1-S3. As a control, we obtained blood samples from 16 healthy subjects from the blood bank (Etablissement Français du Sang): 9 vaccinated individuals (35 to 59 years old) and 7 non-vaccinated individuals (all under the age of 28 years). Control spleens were retrieved after splenectomy of non-vaccinated patients (microspherocytosis and physical trauma, 7 and 17 year-old respectively).

Isolation of cells and serum

Spleen samples were recovered in complete medium (RPMI-1640, 100 mU/ml penicillin, 100 µg/ml streptomycin (all from Invitrogen) and 10% fetal calf serum (FCS, HyClone, Logan, UT) after surgery and were stored at 4°C until processing. In some cases, the entire spleen was maintained under physiological perfusion for 6 h before cell isolation, as described by Buffet et al., [START_REF] Buffet | Ex vivo perfusion of human spleens maintains clearing and processing functions[END_REF] with no influence in results. Cell suspensions were obtained by mechanical dissociation in complete medium. Peripheral blood and bone marrow aspirates were collected in heparin or EDTA tubes for cell isolation. In all experiments, mononuclear cells (MNC) from blood, bone marrow aspirates or spleen cell suspensions were isolated by centrifugation on a Ficoll-Hypaque density gradient. Red blood cells were lysed and the remaining cells were washed with 2% FCS in PBS and resuspended in complete medium containing 50 µM β-mercaptoethanol. Cell viability after isolation was determined by counting with Trypan Blue exclusion and only samples with viability exceeding 90% were considered. In the case of patient RTX6 (B cell content below 1%), the splenic preparation was enriched for B cells, using the B cell-negative isolation kit (Dynal-Biotech, Oslo, Norway) according to the manufacturer's recommendations. Such enrichment was performed in order to obtain a measurable value in ELISPOT assay. Serum was prepared by clotting blood collected in SST tubes (BD Diagnosis, Franklin Lakes, NJ) or by treating one volume of plasma with one volume of BC thrombin reagent (Dade Behring, Marburg, Germany) for 10 min at room temperature. Serum was then inactivated by heating for 30 min at 56°C and frozen in aliquots.

Flow cytometry

Cells were routinely labelled with the following antibodies: fluorescein isothiocyanate (FITC)-conjugated rabbit F(ab') 2 anti-human IgG (F0056) or the corresponding isotype control (X0929) from Dako (Glostrup, Denmark), phycoerythrin (PE)-conjugated antihuman CD27 antibody (clone 1A4-CD27) and PE-cyanin 5.1 (PC5)-anti human CD19 antibody (clone J4.119) from Beckman-Coulter (Fullerton, CA), allophycocyanin (APC) anti-CD19 (clone HIB19) from BD Pharmingen (San Diego, CA). When indicated, labelled cells were incubated with 7-AAD (BD Pharmingen) 10 minutes before analysis in order to exclude dead cells. Cells were analyzed on a FACScan or FACSCalibur, with CellQuest Pro software (BD Biosciences, San Diego, CA).

Memory B cell assay

This procedure was adapted from that described by Crotty et al. [START_REF] Crotty | Tracking human antigen-specific memory B cells: a sensitive and generalized ELISPOT system[END_REF] Briefly, PBMC or MNC were plated at 5 x 10 5 cells /ml in 24-well (5 x 10 5 cells) or 6-well (5 x 10 6 cells) plates, in complete culture medium supplemented with 10 ng/ml pokeweed mitogen extract (PWM, batch 1303H, ICN, MP Biomedicals, Aurora, OH), a 1/10,000 dilution of fixed Staphylococcus aureus, Cowan extract (SAC, Sigma-Aldrich, St-Louis, MO) and 6 µg/ml fully phosphothioated CpG (ODN-2006, [START_REF] Hartmann | Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo[END_REF] Proligo-Sigma-Aldrich). Cells were cultured for six days at 37°C, under an atmosphere containing 5% CO 2 . They were then washed and used to seed 96-well plates: from each culture well of 5 x 10 5 cells, 1/5 of the cells were used for IgG ELISPOT and 4/5 were used for MVA or KLH ELISPOT. From culture wells of 5 x 10 6 cells, 1/20 was used for IgG ELISPOT and 19/20 was used for MVA or KLH ELISPOT. Samples were serially diluted in culture medium, in triplicate, before transfer to ELISPOT plates. Multiscreen 96-well filter plates (MSIPS4510, Millipore, Bedford, MA) were coated by incubation overnight at 4°C with 100 µl of 3 x 10 8 MVA pfu /ml, 2.5 µg/ml keyhole limpet hemocyanin (KLH, Pierce Biochemicals) or 10 µg/ml anti-human Ig polyvalent antibody (Caltag Laboratories, Burlingame, CA). ELISPOT was performed with 1 µg/ml biotinylated goat anti-human IgG Fc (H10015, Caltag Laboratories) followed by 5 µg/ml HRP-conjugated Avidin D (Vector Laboratories, Burlingam, CA) and developed using 3-amino-9-ethylcarbazole (Sigma-Aldrich). Spots were counted in wells showing more than 3 specific spots, in an ELISPOT reader, with AID software (version 3.5, Autoimmun Diagnostika GmbH, Germany). When detected, IgG secreting cell numbers from the corresponding unstimulated wells (cells cultivated in complete medium alone) were substracted to the values obtained in stimulated cells. Each experiment was conducted in 3 independent wells and the results are the mean of the 3 ELISPOT values obtained.

Data are presented as the percentage of total IgG + memory B cells specific for vaccinia virus. When no vaccinia virus-specific spot was detectable, a value of <x was assigned, with x corresponding to the frequency obtained if one vaccinia-specific spot was detected at the lowest dilution.

ELISA

Maxisorp plates (Nunc, Roskilde, Denmark) were coated by incubation overnight at 4°C with 10 6 MVA pfu/well. Plates were blocked by incubation with 10% FCS in PBS for 1 h at 37°C. Triplicates of serum samples were serially diluted in PBS-0.05% Tween20 (PBST) and incubated for 1 h at 37°C. Plates were washed four times in PBST, and were then incubated for 1 h at 37°C with peroxidase-conjugated goat anti-human IgG Fc (Nordic Immunology, Tilburg, The Netherlands) at a concentration of 2 µg/ml in PBST. Plates were washed four times in PBST and incubated with OPD (Sigmafast OPD tablets, Sigma-Aldrich) for 10 min at room temperature. The reaction was stopped by adding 1.8 N H 2 SO 4 , and optical density was measured at a wavelength of 490 nm. The determination of serum titer is detailed in Supplemental Figure S1.

Statistical Methods

Non parametric Kruskal-Wallis two-sided score test was used to compare the frequency of vaccinia virus-specific memory B cells between blood and spleen and between controls and splenectomized patients, as well as serum titers of vaccinia virus antibody between the different groups studied. For the 23 vaccinated individuals for whom both parameters were known, we estimated the Spearman coefficient of correlation R 2 between anti-vaccinia virus antibody titers and the frequency of vaccinia virus-specific memory B cells. All analyses were performed using SAS Software 9.1 (SAS Institute, Cary, NC).

Results

Anti-vaccinia virus memory B cells are concentrated in the spleen

We first compared the proportion of memory B cells against vaccinia virus in the blood and spleen several decades after vaccination, in samples obtained from vaccinated (Table 1, V1-V20) or non-vaccinated (Table 1, C1-C9) individuals. The modified vaccinia virus Ankara (MVA) is a replication-deficient strain of vaccinia virus that was tested as a safer vaccine for smallpox vaccination. [START_REF] Meyer | Mapping of deletions in the genome of the highly attenuated vaccinia virus MVA and their influence on virulence[END_REF] It was used throughout this study for the ELISPOT and ELISA assays, to assess cellular and serological anti-smallpox/vaccinia virus memory. Blood or splenic mononuclear cells were analyzed by ELISPOT against MVA, after 6 days of in vitro polyclonal stimulation, as described by Crotty et al. [START_REF] Crotty | Tracking human antigen-specific memory B cells: a sensitive and generalized ELISPOT system[END_REF] We determined the proportion of anti-vaccinia virus-specific B cells as the ratio of anti-MVA IgG + cells to total IgG + cells (Figure 1A). The members of the vaccinated cohort were aged between 31 and 75 years, whereas the non-vaccinated controls were all under the age of 28 years (Table 1). We first confirmed previous results, [START_REF] Crotty | Cutting edge: long-term B cell memory in humans after smallpox vaccination[END_REF] showing that a median of 0.07% of circulating IgG + memory B cells were specific for vaccinia virus in the blood of individuals vaccinated more than 30 years previously, with the values observed lying between 0.01 and 0.16% (Figure 1B; Table 1). The absence of anti-vaccinia virus memory B cells in the non-vaccinated controls indicated an absence of cross-reactive responses induced by closely related viruses. We retrieved splenic fragments, mostly from patients who had undergone surgery for a pancreatic tumor (see description of patients in Supplemental Table S1). Splenic mononuclear cells from nine patients vaccinated at least 30 years ago, and from two nonvaccinated controls (7 and 17 years old) were analyzed with the anti-MVA ELISPOT assay.

The proportion of anti-vaccinia virus memory B cells in the spleen was much higher, at 0.24%, with values ranging from 0.07 to 0.62% (Figure 1B; V10-V18 in Table 1). No vaccinia virus-specific IgG secreting cells were detected in control culture with unstimulated cells either from blood or from spleen. In three patients from whom both splenic and blood samples could be obtained, the proportion of memory B cells in the spleen and blood differed by a factor of three to ten (Figure 1B insert; V10, V13, V18, in Table 1), these differences being observed even in cases in which the frequency of splenic memory B cell was in the lower range (Table 1, V13 and V18).

Variable impact of rituximab-induced B cell depletion on anti-vaccinia virus memory B cell survival

It has been reported that the circulation is depleted of most of its B cells following treatment with rituximab, a B cell-depleting anti-CD20 antibody. However, the extent of this depletion seems to be more variable in lymphoid organs. [START_REF] Vallerskog | Treatment with rituximab affects both the cellular and the humoral arm of the immune system in patients with SLE[END_REF][START_REF] Ramos | The effect of desensitization protocols on human splenic B-cell populations in vivo[END_REF][19] B cell recovery in the blood starts about six months after the end of the treatment, with normal levels reached after 12 to 20 months. We analyzed the blood of five patients treated with rituximab 25 to 39 months previously (Table 2; Figure 1C). No circulating anti-vaccinia virus memory B cells were detected in two patients (RTX3: <0.007%, RTX5: <0.04%), whereas such cells were detected in two others (RTX4, RTX1), albeit at a low frequency (0.01 and 0.04%). Surprisingly, in the last patient (RTX2), memory B cells specific for vaccinia virus were found 26 months after rituximab treatment (RIT; 2 cycles, see description of patients in Supplemental Tables S2) at a frequency much higher (0.32%) than that observed in the blood of normal individuals analyzed several decades after vaccination. An increase in serum B-cell-activating factor (BAFF) concentration has been reported after RIT in some patients. [START_REF] Lavie | Increase of B cell-activating factor of the TNF family (BAFF) after rituximab treatment: insights into a new regulating system of BAFF production[END_REF] Serum BAFF level was normal in patient RTX2 (not shown), leaving us with no -11 -clear explanation for these findings. Thus, most patients treated with rituximab displayed a lower frequency of anti-vaccinia virus memory B cells in their blood.

Some splenic anti-vaccinia virus memory B cells can resist rituximab treatment

Given the highly variable pattern of memory B cell recovery after RIT, and assuming that the spleen could act as a major reservoir for these cells, we decided to determine the frequency of residual memory B cells in the spleen after RIT. We focused on three cases of idiopathic thrombocytopenic purpura (ITP) who underwent splenectomy after a failure to respond to treatment. One patient (RTX6, Table 2) was splenectomized three months after RIT. No B cells were detected in his blood and the percentage of B cells in the spleen was very low (around 0.1%). These cells were found to be almost exclusively IgG + CD27 + (Figure 2), thus suggesting a memory phenotype. After in vitro stimulation, anti-vaccinia virus memory B cells were detected, amounting to 0.14% of total IgG + B cells (Table 2).

The second patient (RTX7, Table 2) underwent splenectomy six months after the end of RIT. B cells were beginning to reappear in his blood (1.6%, versus 10% in controls) and spleen (10% versus 40% in controls). Memory B cells against vaccinia virus were not detectable in his blood and amounted to 0.06% in his spleen. In the third patient (RTX8, Table 2) in which the spleen was retrieved 15 months after the end of RIT, B cells recovered to normal values in blood and spleen (Supplemental Table S2) but vaccinia virus-specific memory B cells could only be found in the spleen at a frequency of 0.06% of total IgG secreting cells. These three cases evoke that a small pool of splenic anti-vaccinia virus memory B cells (a few tens of thousands memory B cells) have resisted to RIT along with other IgG + switched B cells. As the spleen replenishes with newly formed IgG + B cells, this pool may tend to dilute itself.

No significative correlation between vaccinia virus-specific serum antibody titers and blood memory B cells

We estimated the correlation between the proportion of memory B cells and the level of specific antibodies against vaccinia virus, by using ELISA to determine serum anti-vaccinia virus antibody titers in the blood of most of the subjects analyzed in this study (Tables 1 and2). A large proportion of the patients vaccinated more than 30 years ago had measurable levels of anti-vaccinia virus antibodies, consistent with the findings of previous reports. [START_REF] Crotty | Cutting edge: long-term B cell memory in humans after smallpox vaccination[END_REF][START_REF] Hammarlund | Duration of antiviral immunity after smallpox vaccination[END_REF][START_REF] Amanna | Duration of humoral immunity to common viral and vaccine antigens[END_REF] We took a value of 0.075, the median value for non-vaccinated individuals, as the threshold below which levels of IgG against vaccinia virus were considered negative (Table 1 and Supplemental Figure S1). Three patients with no anti-vaccinia virus antibodies were found to have vaccinia virus-specific memory B cells in their blood (V6, Table 1 and RTX4, SPL5, Table 2). Conversely, in three patients with a reasonably high level of antivaccinia virus antibodies, no circulating anti-vaccinia virus memory B cells could be detected in their blood (V13, Table 1 and SPL4, RTX3, Table 2). No significative correlation was found in this study between the proportion of memory B cells and the titer of antibodies specific for vaccinia virus (Figure 3, for n=23 individuals, R 2 =-0.03, p-value=0.88).

Discussion

We studied the persistence of vaccinia virus-specific memory B cells in the blood and spleen of normal individuals and of patients who had undergone B cell-targeted immune interventions, splenectomy or anti-CD20 mediated B cell depletion.

In individuals vaccinated with the vaccinia virus during their childhood (i.e., more than 30 years before this study) about 0.24% of splenic IgG + B cells recognize this pathogen. This proportion is significantly higher than that for the blood (about 0.07%, consistent with previous reports [START_REF] Crotty | Cutting edge: long-term B cell memory in humans after smallpox vaccination[END_REF][START_REF] Amanna | Duration of humoral immunity to common viral and vaccine antigens[END_REF] ). Taking into account the size of the blood and splenic B cell compartments (approximately 5x10 8 and 4x10 10 B cells respectively, with 10 to 20% IgG + B cells), there are, on average, around 0.5 to 1 x 10 5 memory B cells against vaccinia virus circulating in the blood of vaccinated individuals, and 1 to 2 x 10 7 such cells in the spleen.

Assuming that all pathogens display similar numbers of immunodominant B cell epitopes, thereby engaging similar numbers of memory B cells, this suggests that the splenic B cell compartment has an overall memory against 10 2 to 10 3 different pathogens. The values proposed for the repertoire of long-lived plasma cells in humans are of the same order of magnitude. [START_REF] Hofer | Adaptation of humoral memory[END_REF] It has been suggested that some long-lived plasma cells may be regularly replaced by newly formed plasmablasts, although it remains unclear how this replacement takes place. [START_REF] Odendahl | Generation of migratory antigen-specific plasma blasts and mobilization of resident plasma cells in a secondary immune response[END_REF] A similar turnover may occur for memory B cells, but again, we know little of the basic mechanism of memory formation and the signals conferring on these cells a shorter or longer existence within the individual.

We could not study lymph nodes or Peyer's patches from vaccinated individuals, raising questions about the possibility of other secondary lymphoid organs constituting an additional reservoir for long-lived memory B cells. There was a severe reduction, although not a complete loss, of circulating anti-vaccinia virus memory B cells in most splenectomized patients. This suggests that these cells survive as such in the blood or that there are other accessory niches outside the spleen. Rituximab, a chimeric human/mouse anti-CD20 antibody, has become a major treatment for B cell-related lymphomas and autoimmune disorders. Two to four years after RIT, most patients presented much lower levels of circulating anti-vaccinia virus memory B cells. Analysis of the spleen a few months after RIT showed the presence of residual B cells, with the IgG + phenotype only, -15 -including a small proportion of long-lived memory B cells, despite the total absence of circulating B cells. Strikingly, a low level of circulating anti-vaccinia virus memory B cells was also observed in one patient, three years after a splenectomy performed 2 months after the completion of rituximab treatment. Since B cell depletion was still complete in the blood at the time of splenectomy, this unique case strengthens the view that there are other storage sites for memory B cells, but that these sites do not seem to compensate quantitatively for the absence of the spleen. Bone marrow has been identified as a preferential niche for long-lived plasma cells and for some memory T and B cells. [START_REF] Manz | Lifetime of plasma cells in the bone marrow[END_REF][START_REF] Slifka | Humoral immunity due to long-lived plasma cells[END_REF][START_REF] Paramithiotis | Memory B lymphocytes migrate to bone marrow in humans[END_REF][START_REF] Rosa | The bone marrow: a nest for migratory memory T cells[END_REF] We did not detect any specific enrichment of vaccinia virus memory B cells in the two bone marrow samples that we have analyzed. Overall, these results suggest that the spleen is a major niche for long-lived memory B cells but that despite drastic B cell depleting treatment such as RIT or splenectomy, these cells can slowly reappear in the blood. These treatments therefore do not seem to result in the systematic eradication of the B cell memory carried by these patients, notably the one related to childhood vaccinations.

Consistent with this interpretation, the response to recall antigens seemed to be strongly decreased by rituximab treatment in one study of lymphoma patients, [START_REF] Van Der Kolk | Rituximab treatment results in impaired secondary humoral immune responsiveness[END_REF] whereas this response was found to be preserved in another study. [START_REF] Bearden | Rituximab inhibits the in vivo primary and secondary antibody response to a neoantigen, bacteriophage phiX174[END_REF] The concept of two layers of long-term humoral memory sustained by the existence of long-lived memory B cells and long-lived plasma cells emerged some years ago [START_REF] Manz | Lifetime of plasma cells in the bone marrow[END_REF][START_REF] Slifka | Humoral immunity due to long-lived plasma cells[END_REF] and has subsequently been strongly supported by experimental data. [START_REF] Radbruch | Competence and competition: the challenge of becoming a long-lived plasma cell[END_REF] It has also been proposed that the maintenance of antibodies several decades after a vaccination or an infection was due to a constant production by non specific stimulation of memory B cells. [START_REF] Bernasconi | Maintenance of serological memory by polyclonal activation of human memory B cells[END_REF] In the later case, there should be a quantitative correlation between the frequency of memory B cells and the level of serum antibodies. In several reports in which these parameters were analyzed after vaccination or natural infections, the strength of the correlation varied considerably, [START_REF] Crotty | Cutting edge: long-term B cell memory in humans after smallpox vaccination[END_REF][START_REF] Amanna | Duration of humoral immunity to common viral and vaccine antigens[END_REF][START_REF] Leyendeckers | Correlation analysis between frequencies of circulating antigen-specific IgG-bearing memory B cells and serum titers of antigen-specific IgG[END_REF][START_REF] Nanan | Acute and long-term effects of booster immunisation on frequencies of antigen-specific memory B-lymphocytes[END_REF][START_REF] Quinn | Immune responses to Bacillus anthracis protective antigen in patients with bioterrorism-related cutaneous or inhalation anthrax[END_REF] suggesting that bystander stimulation of memory B cells into short lived plasma cells was unlikely to be a general mechanism. In the present study, in which it was possible to analyze more strongly contrasting situations, no correlation was observed for the 23 patients for whom both serum titers of anti-vaccinia virus antibodies and memory B cells could be determined. In fact, a complete dissociation was observed in several cases, with anti-vaccinia virus antibodies detected in the total absence of the corresponding memory B cells, or memory B cells being present in the absence of the corresponding antibodies. This observation gives additional support to the proposal that long-lived memory B cells and long-lived plasma cells in humans are independent entities, with long-lived plasma cells able to survive for several decades independently of the presence of memory B cells of the same specificity. [START_REF] Amanna | Duration of humoral immunity to common viral and vaccine antigens[END_REF][START_REF] Slifka | Humoral immunity due to long-lived plasma cells[END_REF][START_REF] Manz | Survival of long-lived plasma cells is independent of antigen[END_REF] Nevertheless, B cell memory does not seem to be a uniform phenomenon and one can not exclude that some antigen-specific or non specific stimulation of long-lived memory B cell could also contribute to the production of antibodies. [START_REF] Bernasconi | Maintenance of serological memory by polyclonal activation of human memory B cells[END_REF][START_REF] Zinkernagel | On natural and artificial vaccinations[END_REF] Many unanswered questions remain concerning the homeostatic regulation of long-lived memory B cells. If the spleen is indeed, as proposed here, a main reservoir for these cells, then most memory B cells may only react to a specific recall pathogen once it reaches the spleen, whereas the circulating memory B cells and neutralizing antibodies will provide a first line of defense at the beginning of infection. antigens. Blood and spleen samples from patient V10 (Table 1). Shown is one representative ELISPOT well out of three, corresponding to the indicated dilution for each antigen. (B) Frequency of anti-vaccinia virus memory B cells in blood and spleen, determined by ELISPOT assay. The insert shows data from three patients (V10, V13, V18, Table 1) for whom both blood and spleen were analyzed.

Figure 1 .

 1 Figure 1. Vaccinia virus-specific memory B cells are enriched in the spleen and

  (C) Frequency of anti-vaccinia virus memory B cells determined by ELISPOT assay in blood of splenectomized (SPL) and rituximab-treated (RTX) patients, compared to healthy donors (Control, same samples as in panel B). Bold lines indicate the median values for groups with sample size >5. Dashed symbols correspond to maximum estimates for individuals for whom specific memory B cells were undetectable (see Materials and Methods). p are determined by a non parametric Kruskal-Wallis two-sided test.

Figure 2 .

 2 Figure 2. Rituximab-resistant splenic B cells are CD27 + IgG + .

Figure 3 .

 3 Figure 3. No correlation between anti-vaccinia virus antibodies and memory B cells in

Figure 1 Figure 2 Figure 3

 123 Figure 1

Table 1 . Serum anti-vaccinia virus antibodies and anti-vaccinia virus memory B cells in blood, spleen and bone marrow % vaccinia virus-specific memory B cells

 1

	Individual	Age	Anti-vaccinia
		(years)	virus IgG
			(serum dilution			In bone
			ratio) †	In blood	In spleen	marrow
	Non-vaccinated C1	28	0.22	<0.001 *
	C2	27	0.07	<0.001 *
	C3	24	nd	<0.013 *
	C4	24	0.04	<0.030 *
	C5	23	0.12	<0.010 *
	C6	20	0.08	<0.004 *
	C7	20	0.05	<0.010 *
	C8	17	nd	<0.018 *	<0.002 *
	C9	7	nd	nd	<0.005 *
	Median		0.075
	Vaccinated
	V1	59	0.27	0.037
	V2	56	0.28	0.110
	V3	53	1	0.088
	V4	53	0.38	0.112
	V5	53	0.10	0.134
	V6	48	0.07	0.062
	V7	48	0.49	0.010
	V8	45	nd	0.117
	V9	35	0.73	0.155
	V10	75	1.16	0.047	0.456
	V11	74	0.53	nd	0.157
	V12	66	ni	nd	0.309
	V13	59	0.40	<0.012 *	0.068
	V14	58	0.29	nd	0.301
	V15	57	nd	nd	0.616
	V16	53	nd	nd	0.230
	V17	45	0.21	nd	0.238
	V18	31	ni	0.031	0.088
	V19	53	0.68	0.022		0.038
	V20	47	0.10	0.067		0.018
	Median		0.380	0.067	0.238

*

Maximum estimate for individuals for whom memory B cells were undetectable (see the Methods section), †Dilution factor with respect to the positive control, see definition in the Materials and Methods section, nd: not done, ni: not interpretable (patient receiving intravenous Ig at the time of sample collection).

Table 2 . Serum anti-vaccinia virus antibodies and anti-vaccinia virus memory B cells in rituximab-treated (RTX) and splenectomized (SPL) patients % vaccinia virus-specific memory B cells

 2

	Patient	Age at	Time after	Anti-vaccinia
		sample	rituximab	virus IgG
		collection	(months)	(serum dilution	In blood	In spleen
				ratio)
	RTX1	83	39	0.12	0.044
	RTX2	61	26	0.84	0.317
	RTX3	55	29	0.32	<0.007 *
	RTX4	45	25	0.05	0.010
	RTX5	42	32	0.25	<0.041 *
	RTX6	79	3	ni	- †	0.140
	RTX7	70	6	ni	- †	0.063
	RTX8 RTX9	62 67	15 2 + 36 ≠	0.47 0.40	- † 0.015	0.064
	Patient	Age at	Years after	Anti-vaccinia	% vaccinia virus-specific
		sample	splenectomy	virus IgG	memory B cells in blood
		collection		(serum dilution
				ratio)
	SPL1	60	50	1.10	0.010
	SPL2	59	44	0.20	0.041
	SPL3	52	7	0.34	0.009
	SPL4	46	35	0.28	<0.009 *
	SPL5	35	30	0.01	0.062

*

Maximum estimate for individuals for whom memory B cells were undetectable. †B cell depletion precluded blood analyses. ≠ Patient was splenectomized 2 months after rituximab treatment. Blood was collected 36 months after splenectomy. Other definitions as in Table

1

, see clinical description in Supplemental Tables

S2-S3

.

Acknowledgments

We thank Drs. B. Ranque and A. Alcaïs for statistical analysis, S. Arguès for assistance with ELISA, Dr. Puydupin from the EFS St-Vincent de Paul for providing blood samples and Dr. Alyanakian from Hôpital Necker-Enfants Malades for serological determinations. We thank Q. Gueranger for helpful suggestions, and L. Abel and J.L. Casanova for critical reading of the manuscript. This work was supported by the Fondation Princesse Grace, the Agence Nationale pour la Recherche ("B cell memory", MIIM program) and the Ligue Contre le Cancer (Equipe labellisée). M.M.-M. was supported by successive fellowships from the Fondation de France, the Fondation Singer-Polignac and the Région Ile-de-France. C.A.R. is a Research Director at the Centre National de la Recherche Scientifique.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Abstract

The fact that you can vaccinate a child at 5 years of age and find lymphoid B cells and antibodies specific for this vaccination 70 years later remains an immunological enigma. It has never been determined how these long-lived memory B cells are maintained and whether they are protected by storage in a special niche. We report that, whereas blood and spleen compartments present similar frequencies of IgG + cells, anti-smallpox memory B cells are specifically enriched in the spleen where they account for 0.24% of all IgG + cells, i.e. 10-20 million cells, more than 30 years after vaccination. They represent in contrast only 0.07% of circulating IgG + B cells in blood, i.e. 50-100,000 cells. An analysis of patients either splenectomized or rituximab-treated confirmed that the spleen is a major reservoir for long-lived memory B cells. No significant correlation was observed between the abundance of these cells in blood and serum titers of anti-vaccinia virus antibodies in this study, including in the contrasted cases of B-cell depleting treatments. Altogether, these data provide evidence that in humans, the two arms of B cell memory -long-lived memory B cells and plasma cells-have specific anatomical distributions -spleen and bone marrow-and homeostatic regulation.

Splenectomy affects the circulating long-lived memory B cell pool

If long-lived memory B cells are principally maintained in the spleen, then the frequency of these cells should be significantly lower in patients that have been splenectomized several years ago. We collected blood samples from five patients, who had undergone splenectomy 7 to 50 years previously (see description of patients in supplemental Table S3). The levels of circulating anti-vaccinia virus memory B cells were all below the median of the control values (Figure 1C; Table 2, SPL4: no detectable anti-vaccinia virus memory B cells, SPL1: 0.01%, SPL3: 0.009%, SPL2, for which small accessory spleens were detected: 0.04% and SPL5: 0.06%). These results suggest that splenectomy may lead to a consistent loss of long-lived memory B cells and that this deficiency can persist even several decades after the surgical procedure. Nevertheless, these cells can still be found, albeit at diminished levels, in the blood of most of these patients, suggesting the existence of other minor storage sites outside the spleen.

Further evidence to support this hypothesis is provided by the case of a rituximab-treated patient who underwent splenectomy two months after four cycles of RIT and who was analyzed 36 months after splenectomy. A small proportion of circulating anti-vaccinia virus memory B cells could be detected in this patient (RTX9: 0.02%, Table 2).

In two cases in which it was possible to analyze a bone marrow sample not contaminated with malignant cells or blood, we detected no significant enrichment of anti-vaccinia virus memory B cells over average values for blood (V19: 0.04%, V20: 0.02%, Table 1).

Author contributions


Ahmed Zouari

Roua Lajnaf

Christelle Lopez

Pierre Schuck

Hamadi Attia

Mohamed Ali Ayadi

Physicochemical, techno-functional, and fat melting properties of spray-dried camel and bovine milk powders

Keywords: spray drying, powder, dromedary milk, foam capacity, foaming, rehydration

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

A wide range of foods and food ingredients are commercialized in the form of powders. Indeed, powder systems offer several advantages to consumers and manufacturers, such as the shelf-life extension, the packaging and transport costs reduction, and manutention easiness. Actually, food powders (e.g., milk powders) are often produced using the spray-drying technology. This technique allows the production of milk powders with acceptable nutritional and functional properties [START_REF] Schuck | Functionality of milk powders and milk-based powders for end use applications-A review[END_REF]. Most of the quality indicators of milk powders are related to their physicochemical composition (e.g., water activity and free fat amount), as well as their physical (e.g., particle size), techno-functional (e.g., rehydration and foaming), and sensorial (e.g., color and odor) properties.

During spray drying, water is evaporated in a brief heat exposure to dried-warm air. This exposure can modify the structure and functionalities of some heat-sensitive milk proteins [START_REF] Zouari | Changes in physical and biochemical properties of spray dried camel and bovine milk powders[END_REF]. The proteins denaturation and their subsequent aggregation may result in the loss of their functionality JFDS-2020-0672 Submitted 4/24/2020, Accepted 11/8/2020. Authors Zouari, Lajnaf, Attia, and Ayadi are with Valuation, Security and Food Analysis Laboratory, National Engineering School of Sfax, Sfax University, Sfax, Tunisia. Authors Zouari, Lopez, and Schuck are with INRAE, STLO, Rennes, F-35000, France. Direct enquiries to author Zouari (E-mail: ahmedzouarri@gmail.com).

especially their rehydration and color properties [START_REF] Haque | Change in molecular structure and dynamics of protein in milk protein concentrate powder upon ageing by solid-state carbon NMR[END_REF][START_REF] Schuck | Functionality of milk powders and milk-based powders for end use applications-A review[END_REF]. The rehydration properties (i.e., solubility, dispersibility, and wettability) describe the reconstitution process of milk powders in water. Milk powders with reduced rehydration properties may impair their sensorial acceptability and techno-functional properties. One of the most important techno-functional properties of milk powders is the foaming properties. These properties are related to the interfacial properties of milk proteins, as well as their capacity to adsorb to the water-air interface.

Nowadays, the consumption spectra of camel milk is becoming wider and wider especially in many nonregular consuming countries. In general, camel and bovine milks present similar proximate composition. The proteins fraction of camel and bovine milks is essentially composed of caseins (around 80%) and whey proteins (around 20%) [START_REF] Felfoul | Proteomic profiling of camel and cow milk proteins under heat treatment[END_REF][START_REF] Omar | Quantification of major camel milk proteins by capillary electrophoresis[END_REF]. Actually, it was acknowledged that the camel milk proteins contain higher β-casein (47%) and lower ᴋ-casein (3.5%) content than those of bovine milk [START_REF] Omar | Quantification of major camel milk proteins by capillary electrophoresis[END_REF][START_REF] Zouari | Acid gelation of raw and reconstituted spray-dried dromedary milk: A dynamic approach of gel structuring[END_REF]. Besides, the composition of bovine milk whey proteins showed a predominance of the β-lactoglobulin (β-Lg) and the α-lactalbumin (α-La). The particularity of camel milk whey is the lack of β-Lg, overexpression of α-La, and the presence of some specific proteins, such as peptidoglycan recognition protein (PGRP) [START_REF] Felfoul | Proteomic profiling of camel and cow milk proteins under heat treatment[END_REF][START_REF] Kappeler | Expression of the peptidoglycan recognition protein, PGRP, in the lactating mammary gland[END_REF][START_REF] Lajnaf | The foaming properties of camel and bovine whey: The impact of pH and heat treatment[END_REF].

Recent studies on camel milk powder have been focused on its particle structure and surface composition (Zouari, Schuck et al., 2020), as well as its behavior during drying [START_REF] Habtegebriel | Effect of operating parameters on the surface and physico-chemical properties of spray-dried camel milk powders[END_REF][START_REF] Zouari | Effect of outlet drying temperature and milk fat content on the physicochemical characteristics of spray-dried camel milk powder[END_REF][START_REF] Zouari | Effect of spraydrying parameters on the solubility and the bulk density of camel milk powder: A response surface methodology approach[END_REF] and its physicochemical changes upon storage [START_REF] Ho | Changes in physicochemical properties of spray-dried camel milk powder over accelerated storage[END_REF][START_REF] Zouari | Changes in physical and biochemical properties of spray dried camel and bovine milk powders[END_REF]. However, none of these explained the practical conditions to use skimmed or whole-fat camel milk powder in future applications. In this context,the objective of this study was to appraise the physicochemical characteristics of camel milk powders at different inlet\oulet drying temperatures (160ࢨ80 °C, 180ࢨ80 °C, and 200ࢨ80 °C). The free fat content, the loose and tapped bulk densities, the particles size distribution, and the rehydration properties (insolubility, dispersibility, and wettability indexes) were investigated. Then, the melting behavior of camel milk fat (anhydrous form and within the powder) was evaluated. Afterward, the SDS-PAGE was used to evaluate the denaturation extent of spray drying temperature on camel milk proteins. Finally, the foaming capacity (FC) and stability (FS) of skimmed (before drying) and reconstituted camel milks (after drying) were determined. To create a comparative study, we have also produced and evaluated bovine milk powders following the same drying conditions and the same evaluation techniques.

MATERIALS AND METHODS

Milk samples

Fresh whole-fat camel (Camelus dromedarius) and bovine (Bos taurus) milks were collected from two separated farms in southern Tunisia (Gabes and Sfax governorates, respectively). Milk samples were immediately stabilized against microbiological development with the addition of 0.02% (w/w) of sodium azide and were transported to the laboratory within 2 hr after milking. Thereafter, skimmed bovine and camel milks were obtained following one or three successive centrifugations (2,000 g, 15 min, and 5 °C), respectively. The fat contents of whole-fat and skimmed camel and bovine milks were analyzed using Gerber method [START_REF] Kleyn | Determination of fat in raw and processed milks by the Gerber method: Collaborative study[END_REF].

Drying conditions

Camel and bovine milk samples, either whole-fat (27 g/L) or skimmed (<1 g/L),were spray-dried using a single stage mini spray dryer configured in cocurrent system (B-290 Büchi Labortechnik AG, Flawil, Switzerland). Each milk was introduced to the sprayer at 20 °C. During all experiments, the absolute humidity of the inlet air was equal to 5 g of water per kg of dry air. The average droplets residence time in the drying chamber was equal to 1 s (experimentally determined). The rates of both drying air flow (35 m 3 /hr) and pressurized air flow at the two-fluid atomizer nozzle (47 m 3 /hr , corresponding to a pressure of 0.41 bar) were held constant. The milk feed rate was equal to 0.89 ± 0.12 kg/hr. Under these drying conditions, three skimmed camel or bovine milk powders (unique trials) were produced using 160ࢨ80 °C, 180ࢨ80 °C, and 200ࢨ80 °C as drying temperatures. The whole-fat camel or bovine milk powders were only produced using 200ࢨ80 °C as drying temperatures. The outlet drying temperature (80 °C) was maintained by slightly managing the feed rate. The standard deviation for all temperatures was equal to 2 °C. At the end of the drying process, the powders were collected in sterilized glass vessel and placed at 4 °C until use.

Physicochemical characterization of the powders

The water activity of the produced powders was determined using an a w -meter (Novasina RTD 200/0 and RTD 33,Pfäffikon,Switzerland). Camel and bovine milk powder compositions were determined as described by [START_REF] Schuck | Functionality of milk powders and milk-based powders for end use applications-A review[END_REF].

The free fat content was measured by mixing 10 g of camel or bovine milk powders for 5 min with 50 mL of petroleum ether [START_REF] Schuck | Functionality of milk powders and milk-based powders for end use applications-A review[END_REF]. The mixture was filtered through Whatman filter paper (Whatman N°40, Maidstone, UK). Then, the obtained filtrate was evaporated at 60 °C under vacuum to remove the petroleum ether. The free fat content (expressed in g/100 g of total fat of the analyzed powder) was measured using the following equation:

Free fat content =

Weight o f fat a fter evaporation Total fat o f the analyzed powder × 1000

Densities and size distribution of powder particles

The loose bulk density and the tapped bulk density (150 tapes) of the produced powders were measured as described by [START_REF] Zouari | Effect of outlet drying temperature and milk fat content on the physicochemical characteristics of spray-dried camel milk powder[END_REF] using a PT-N powder characteristics tester (Hosokawa Micron, Osaka, Japan).

The size distributions of skimmed camel and bovine milk powder particles were determined using a laser light scattering Mastersizer 2000 (Malvern Instruments Ltd., Malvern, UK), equipped with a dry powder feeder (Scirocco 2000, Malvern Instruments, Worchestershire, UK). The feeder was operating with a dispersion air pressure of 4 bars. The d 50 and the span (defined below) were chosen as size distribution indicators.

span = d 90 -d 10 d 50
where d 10 , d 50 , and d 90 represent the particle size of the produced powders lying below 10, 50, and 90% of the whole distribution, respectively.

Rehydration ability of the powders

The rehydration ability of the produced powders was determined by considering the insolubility, wettability, and dispersibility indexes (DIs) as described by [START_REF] Schuck | Functionality of milk powders and milk-based powders for end use applications-A review[END_REF]. Ten grams of each skimmed milk powder were vigorously mixed using a laboratory blender (4,000 rpm) with 100 mL of MiliQ water and antifoaming agent (Octanol, 2 to 3 drops) at 20 °C during 90 s. Thereafter, the mixture was centrifuged at 160 g during 5 min. The sediment volume (expressed in mL) was recorded, which corresponded to the insolubility index.

The wettability time reflects the necessary time (expressed in min) to allow the total penetration of 10 g of camel and bovine milk powders in 100 mL of MiliQ water at 20 °C without disruption. After sinking, the wetted milk powder was then mixed with water using a glass spatula during 15 s. The resulting mixture was filtered using a sieve of 200 μm mesh size. The dry matter of the filtrate was then estimated by drying the filtrate at 105 °C for 7 hr in an oven. The DI was then determined following the below formula:

DI = (W + w) .X DM w. 100-XRW 100
where W is water weight, w is powder weight, X DM is dry matter of the filtrate (%, w/w), and X RW is residual free water of the powder (%, w/w).

Food Engineering, Materials Science, & Nanotechnology

Camel milk powder characteristics… 2.6 Denaturation extent of camel and bovine proteins 2.6.1 Whey protein nitrogen index. The noncasein nitrogen (NCN) and the nonprotein nitrogen (NPN) were determined as described by [START_REF] Schuck | Functionality of milk powders and milk-based powders for end use applications-A review[END_REF]. The whey protein nitrogen index (WPNI) was calculated as follows:

WPNI g N 2 k g-1 = NCN 6.25 - NPN 6.19 × 10
2.6.2 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 15% of polyacrylamide under reducing conditions as described by [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF] with slight modifications. Skimmed and reconstituted camel and bovine milks were first diluted with ultrapure water (2:1, v/v). Then, 5 μL of diluted of each milk was added to 5 μL of ultrapure water and 5 μL of buffer solution (SDS: 10% (w/w); 0.5 M β-mercaptoethanol; 0.5 M tris-HCl (pH = 6.8); glycerol 2% (w/w); and bromophenol blue 0.1% (w/w). Afterward, the mixture was heated for 4 min at 95 °C.

The electrophoresis analysis was carried out using a Mini-Protean Tetra Cell (Bio-Rad Laboratories, Hercules, CA, USA) with a constant voltage of 120 Volt during 2 hr. After migration, proteins were detected by staining the gel for at least 30 min with a coloring mixture composed of Coomassie Blue R-250 (0.1%,v/v), acetic acid (80%),and ethanol (96%).Finally,the gel was placed into two successive discoloration solutions (acetic acid (80%), ethanol (96%), and ultrapure water). The approximate molecular weight of each milk protein was evaluated using CLIQS Software (Version 1.1, TotalLab Ltd, Newcastle upon Tyne, UK) calibrated with molecular weights of known proteins (10 to 100 kDa, Promega Corporation, Durham, NC, USA).

Differential scanning calorimetry

The thermal properties of camel and bovine milk fat within the powders and in their anhydrous state were investigated using a differential scanning calorimeter (TA Q-1000, TA Instruments, Eschborn, Germany). The calibration of the DSC system was achieved with Indium (melting point 156.6 °C). About 10 mg of each powder sample was sealed in aluminum hermetic pans. The behavior of fat within whole-fat milk powders was recorded from 0 to 65 °C by increasing the temperature with a rate of 1 °C/min. The anhydrous fat fractions of whole-fat camel and bovine milk powders were obtained by extracting the total fat (with hexane/isopropanol [3:1, v:v]). The melting profiles of these fractions were recorded from -30 to 65 °C with a rate of 1 °C/min. The TA Universal Analysis 2000 software (Ta instruments, Waters LLC) was used to determine the melting characteristics of all analyzed fractions.

Foaming properties

The FC and stability of skimmed (before drying) and reconstituted (after drying) camel and bovine milk powder were determined as described by [START_REF] Lajnaf | The foaming properties of camel and bovine whey: The impact of pH and heat treatment[END_REF] with slight modifications. Ten milliliters (V 0 , expressed in mL) of each sample (proteins concentration 0.1%, w/v) were whipped (at 25 °C) in a graduated container (length 84 mm; diameter 38 mm) using an ULTRA-TURAX T 25 basic (IKA WERKE, Germany) at a rapid speed (13,500 rpm) for 2 min. The volume of whipped milks (V 1 , ex-pressed in mL) was immediately recorded and used to calculate the FC following the below equation:

FC (%) = V 1 -V 0 V 0 × 100
The foaming stability (FS) is defined as the necessary duration (expressed in seconds) to reduce the total foam volume by 50%.

Statistical analysis

All performed analyses and measurements in this work were carried out in triplicate. The statistical differences were examined using SPSS 19 software following the Student's t-test, one-way ANOVA, and Tukey post-hoc test with a confidence level of 95%.

RESULTS AND DISCUSSION

3.1 Physicochemical characteristics 3.1.1 Composition of the milks and powders. The compositions of whole-fat and skimmed camel or bovine milks are presented in Table 1. The results indicated that, whether skimmed or not, camel and bovine milks presented similar total solids, total protein, lactose, and fat contents (P > 0.05, Table 1). However, both whole-fat and skimmed bovine milks contained significantly higher casein amounts as compared with camel milks (P < 0.05, Table 1). It is important to note that whole-fat and skimmed camel milks presented higher whey protein and ash contents compared to bovine milks (P < 0.05, Table 1). These differences are in agreement with several other studies [START_REF] Felfoul | Deposit generation during camel and cow milk heating: Microstructure and chemical composition[END_REF][START_REF] Konuspayeva | The composition of camel milk : A meta-analysis of the literature data[END_REF][START_REF] Zouari | Changes in physical and biochemical properties of spray dried camel and bovine milk powders[END_REF]). The latter studies indicated that the observed differences in camel and bovine milk composition mainly depend on the feeding characteristics and the water availability.

The physicochemical compositions of camel and bovine milk powders are presented in Table 1. The water activity of both skimmed and whole-fat milk powders slightly ranged from 0.24 to 0.25. This observation indicated that the fat did not interact with water during water activity measurement. Actually, it was demonstrated that fats were considered as nonreacting component with water and had no influence on the water activity of milk powders [START_REF] Jouppila | Glass transition, water plasticization, and lactose crystallization in skim milk powder[END_REF]Zouari, Schuck et al., 2020). In addition, [START_REF] Schuck | Water activity and glass transition in dairy ingredients[END_REF] indicated that the water activity of nonfat and high-fat milk powders is mainly controlled by the moisture content of nonfat solids (i.e., proteins, lactose, and minerals). For all drying conditions, the total protein and the caseins fraction of bovine milk powders were significantly higher than those of camel milk powders (P < 0.05, Table 1). Moreover, the whey protein fraction of camel milk powders was significantly higher than that of bovine milk powders (P > 0.05, Table 1). Likewise, camel milk powders contained higher ash and lower lactose quantities (P < 0.05, Table 1). Moreover, both milk powders showed the same fat content (skimmed: 1 g/100 g and whole-fat: 20.0 g/100 g). However, the free fat content in camel milk powders was in all cases lower than that observed in bovine milk powders (P < 0.05, Table 2). Similar observations were reported by Zouari, Schuck et al. (2020). The latter indicated that camel milk fat was homogeneously distributed in the camel milk powder showing higher encapsulation degree. This was linked to the smaller mean diameter of camel milk fat globules compared to bovine milk fat globules [START_REF] Karray | La matière grasse du lait de dromadaire: Composition, microstructure et polymorphisme[END_REF] and the higher mechanical resistance to disruption of camel milk fat globules as compared to those of bovine ones (Zouari, Schuck et al., 2020). These milk fat globule characteristics limited the free fat exposure to the camel milk powder surface.

Size distribution and densities of powder particles.

The loose bulk density and the tapped bulk density of the produced camel and bovine milk powders are presented in Table 2. The highest loose bulk density and the tapped bulk density were recorded for camel milk powders as compared to those of bovine milk powders (P < 0.05, Table 2). Similar observations were previously reported by [START_REF] Habtegebriel | Effect of operating parameters on the surface and physico-chemical properties of spray-dried camel milk powders[END_REF] and [START_REF] Zouari | Effect of spraydrying parameters on the solubility and the bulk density of camel milk powder: A response surface methodology approach[END_REF]. These authors reported that camel milk powders presented higher bulk densities than those observed for bovine milk powders. Interestingly, for both milk powders, the loose and tapped bulk densities decreased, while powder fat and free fat contents increased (P < 0.05, Table 2). Kim, Chen, and Pearce (2005a) indicated that the free fat amount impacted the flowability and the loose bulk density of milk powder. Indeed, this author reported that the removal of surface free fat induced an increase in the flowability and the bulk density of the powders. This explained the highest bulk density of camel milk powder as compared to those of bovine ones, since camel milk powder showed the lowest surface free fat content.

In our study, the analysis of the powder particles size distribution highlighted that camel milk powders have significantly lower d 50 than that of bovine milk powders (P < 0.05, Table 2). The width of the size distribution is usually evaluated by measuring the span value. For all produced camel and bovine milk powders, the span value ranged from 1.3 ± 0.1 to 2.1 ± 0.1 for bovine milk powders and from 1.4 ± 0.1 to 1.9 ± 0.1 for camel milk powders (Table 2). Analyses of the d 50 and the span indicated that the produced camel and bovine milk powders presented a narrow size distribution with low d 50 . This indicated that these powders were composed of fine particles. Such kind of powders resulted in high bulk density (Barbosa-Canovas, Ortega-Rivas, Juliano, & Hong, 2005).

Besides, results of this study indicated that by increasing the inlet drying temperature, the d 50 decreased while the span is increasing. This indicated that at high drying temperature, larger particles were produced. Our results are in agreement with Fang, Rogers, Selomulya, and Chen (2012) who reported that the inlet drying temperature affected the powder particle size. Indeed, these authors demonstrated that the particles were larger when dried at high temperatures due to rapid water removal and the high occurrences of particle shrinkage. This resulted in larger particle size (lower d 50 ) and wider size distribution (higher span). 2. This index is widely used in dairy industry to classify the produced powders (from very low heat treatment to high heat treatment) based on the heat sensitivity of some whey proteins [START_REF] Schuck | Functionality of milk powders and milk-based powders for end use applications-A review[END_REF]. In this study, the produced camel or bovine milk powders showed approximately the same WPNI (around 8.5 and 9.6 g of N 2 /kg) regardless of the inlet drying temperature (Table 2). Indeed, it was reported that the outlet drying temperature is the most denaturizing temperature. During drying, the particles temperature quickly reaches the outlet drying temperature (Woo, & Bhandar, 2013), which explained the similarities of the obtained WPNI.

Whey protein nitrogen index. The Whey protein nitrogen index (WPNI) of the produced camel and bovine milk powders is presented in Table

However, we noticed that whole-fat and skimmed camel milk powder were less denaturized than those of bovine ones (P < 0.05, Table 2). This could be related to the whey protein composition of both camel and bovine milks and their heat sensitivity.In fact, [START_REF] Farah | Effect of heat treatment on whey proteins of camel milk[END_REF] analyzed the heat denaturation of camel whey proteins by

Food Engineering, Materials Science, & Nanotechnology

Camel milk powder characteristics…

.3 ± 0.1 a 1.4 ± 0.1 a,b 1.4 ± 0.1 a,b 1.6 ± 0.1 b 1.7 ± 0.1 b 1.9 ± 0.1 b,c 2.1 ± 0.1 c 1.8 ± 0.1 b,c Densities (kg/m)
Loose bulk density 245 ± 2.9 a 301 ± 4.2 b 231 ± 2.9 c 299 ± 2.4 b 257 ± 5.8 d 289 ± 2.6 e 202 ± 1.1 f 231 ± 2.5 c Tapped bulk density 458 ± 5.5 a,d 558 ± 6.1 b 455 ± 1.6 a 558 ± 1.9 b 468 ± 6.9 d 518 ± 5.7 e 378 ± 3.5 f 419 ± 4.2 g Rehydration ability Insolubility Index (mL) 0.5 ± 0. means of WPNI and concluded that camel milk whey proteins had a significantly lower heat sensitivity than those of bovine milk. This explained the highest WPNI of camel milk powders.

Rehydration properties.

The rehydration characteristics of the produced powders are presented in Table 2. The rehydration properties reflect the reconstitution ability of the milk powder in water. Our findings showed that the insolubility indexes of skimmed or whole-fat milk powders ranged from 1 to 0.1 mL, which corresponds to a solubility of 99% to 99.9%. This result was in agreement with the literature data since it was reported that the solubility of spray-dried skimmed bovine milk powder is usually higher than 99% [START_REF] Pugliese | Physical characterization of whole and skim dried milk powders[END_REF]. The highest insolubility index (IS = 1 mL, i.e., a solubility of 99%) was recorded for camel milk powder produced using low air inlet drying temperatures (160ࢨ80 °C).

In addition, skimmed or whole-fat camel and bovine milk powders showed a wettability time higher or close to 30 min for all produced powders. Several studies showed that the wettability is dependent on the surface characteristics and the size distribution of milk powder particles [START_REF] Gaiani | Use of a turbidity sensor to characterize micellar casein powder rehydration: Influence of some technological effects[END_REF][START_REF] Kim | Surface characterization of four industrial spraydried dairy powders in relation to chemical composition, structure and wetting property[END_REF]. In the current study, all measured d 50 of all produced camel and bovine milk powders were lower or closer to 12 μm. Such powder granulometry limited the water penetration inside the milk powder leading to a low powder wettability [START_REF] Gaiani | Use of a turbidity sensor to characterize micellar casein powder rehydration: Influence of some technological effects[END_REF]. Moreover, both milk powders dispersibility values have increased with the intensification of the drying temperature and the decrease of the particles d 50 . The highest DI (DI = 90%) was recorded for both milk powders at the drying temperature of 200ࢨ80 °C. [START_REF] Langrish | An investigation and quantitative assessment of particle shape in milk powders from a laboratory-scale spray dryer[END_REF] have reported that at low inlet air drying temperatures, the produced particles showed a collapsed structure with a high bulk density. This could limit the water penetration to the powder particles, which may lead to a low wettability and dispersibility. Furthermore, it was shown that some technological assessments, such as instantization and agglomeration, are able to enhance the wettability and dispersibility of milk powders. In fact, these techniques reduce the particles bulk density through increasing their size distribution making them porous [START_REF] Cruz | Final drying of whole milk powder in vibrated-fluidized beds[END_REF]. This facilitates the water penetration to the particles and improves the wettability and dispersibility of milk powders. It is important to note that the dispersibility of whole-fat camel and bovine milk powders decreased drastically as compared to the skimmed ones (Table 2). This could be linked to the exposed fat at the powder particle surface, which could limit the powder dispersibility in water (Zouari, Schuck et al., 2020).

For the rest of the study, the drying temperatures of 200ࢨ80 °C were adopted since the resulting powders showed the best rehydration properties (Table 2).

Melting behavior of camel and bovine milk fats within the powder and in anhydrous state

The melting properties of fats mainly depend on their fatty acid and triacylglycerol composition, on the dispersion of the fat (in emulsion droplets or in an anhydrous state) and on the applied thermal kinetics. Figure 1 shows the differential scanning calorimetry (DSC) thermograms recorded on heating of wholefat camel and bovine milk powders and the respective anhydrous fats obtained after extraction. The DSC thermograms recorded for whole fat camel milk powders exhibited three main endotherms corresponding to the successive melting of groups of triacylglycerols dispersed within the lipid droplets, with a final melting point of 46.3 ± 0.2 °C (Figure 1Aa). On heating, the anhydrous camel milk fat exhibited four successive endotherms, with a final melting point of 45.1 ± 0.2 °C (Figure 1Ba). The differences in the final melting points could be attributed to the formation of different crystals. The DSC melting thermograms recorded for camel milk fat are in agreement with previous studies [START_REF] Lopez | Crystallisation and melting properties of dromedary milk fat globules studied by X-ray diffraction and differential scanning calorimetry. Comparison with anhydrous dromedary milk fat[END_REF]. The DSC thermograms recorded for whole fat bovine milk powders and anhydrous bovine milk fat exhibited four main endotherms, with final melting points of 39.8 ± 0.5 °C and 38.8 ± 0.5 °C (Figure 1Ab and Bb). Our study perfectly agrees with the data reported by [START_REF] Kim | Melting characteristics of fat present on the surface of industrial spray-dried dairy powders[END_REF] and [START_REF] Vignolles | Methods' combination to investigate the suprastructure, composition and properties of fat in fat-filled dairy powders[END_REF]. These authors found that the melting temperature significantly decreased from 40.2 °C (for whole-fat bovine milk powder) to 39.8 °C (for anhydrous bovine milk fat) showing four distinct endothermic peaks.

The different melting properties observed between camel milk fat and bovine milk fat, either dispersed within the powder particles or in anhydrous state, are mainly governed by the differences in the fatty acid composition [START_REF] Karray | La matière grasse du lait de dromadaire: Composition, microstructure et polymorphisme[END_REF]. From a technological point of view, the DSC determinations highlighted the fact that the milk fats will be at least partially in a solid state below their final melting temperatures.

Protein analysis

Figure 2 shows the SDS-PAGE patterns of skim (A, C) and reconstituted (B, D) camel and bovine milks, respectively. Under the denaturizing conditions, the SDS-PAGE gives an approximation of the denaturation extent of the spray drying on camel and bovine milk proteins. The first detected bands had a molecular weight ranging from 60 to 100 kDa (Figure 2). These bands were attributed to some whey proteins, of which the latest band corresponded to the serum albumin (Figure 2,bands 5,16,28,and 38). The estimated molecular weight (MW) of this protein was equal to 66 kDa for both camel and bovine milks. In addition, some other whey proteins were detected in skimmed and reconstituted camel milks corresponding to the PGRP (Figure 2,bands 10 and 22,MW = 18 kDa) and α-La (Figure 2, bands 11 and 23, MW = 14 kDa), respectively. In addition, the β-Lg (Figure 2C, bands 32 and 42, MW = 15 kDa) and the α-La (Figure 2, bands 33 and 43, MW = 11 kDa) were identified in bovine milk.Our results highlighted the absence of β-Lg in both skimmed and reconstituted camel milk as already reported by several previous studies [START_REF] El-Hatmi | Characterisation of whey proteins of camel (Camelus dromedarius) milk and colostrum[END_REF][START_REF] Felfoul | Proteomic profiling of camel and cow milk proteins under heat treatment[END_REF][START_REF] Lajnaf | The foaming properties of camel and bovine whey: The impact of pH and heat treatment[END_REF]. It was reported that the β-Lg was the most heat-sensitive protein in bovine milk [START_REF] Corredig | The mechanisms of the heat-induced interaction of whey proteins with casein micelles in milk[END_REF]. Its absence from camel milk further endorsed the highest measured WPNI for camel milk powders.

The camel milk caseins fraction showed comparable proteins composition with that of bovine milk. In fact, some identified bands were attributed to α-casein (Figure 2,bands 7,18,19,30,and 40),bands 6,17,29,and 39), and κ-casein (Figure 2,bands 8,20,31,and 41). For skimmed and reconstituted camel milks, the estimated molecular weights were equal to 35, 27, and 18 kDa for β-casein, α-casein, and κ-casein, respectively. The α s1 -casein (Figure 2, band 18) and α s2 -casein (Figure 2, band 19) were only detected in reconstituted camel milk with molecular weights of 25 and 27 kDa, respectively. Our results are in perfect agreement with several studies [START_REF] Ereifej | Comparison and characterisation of fat and protein composition for camel milk from eight Jordanian locations[END_REF][START_REF] Felfoul | Proteomic profiling of camel and cow milk proteins under heat treatment[END_REF][START_REF] Omar | Quantification of major camel milk proteins by capillary electrophoresis[END_REF].

According to the present SDS-PAGE analysis, there was no disappearance or appearance of new bands between both skimmed and reconstituted bovine and camel milks.This confirmed the high solubility indexes of camel and bovine milk powders (>99.9%). Indeed, it was reported that the alteration of powder solubility is mainly a consequence of milk protein denaturation and/or aggregation through hydrophobic linkages [START_REF] Thomas | Milk powders ageing: Effect on physical and functional properties[END_REF]. At this stage of the study, the SDS-PAGE together with the WPNI analysis indicated that the amount of insoluble material was relatively low to alter the solubility of camel and bovine milk powders.

Foaming properties

Figure 3 illustrates the foaming properties of skimmed (before drying) and reconstituted (after drying) camel and bovine milks. In this study, significant higher foaming properties were recorded for the reconstituted milks as compared to those of skimmed ones (P < 0.05, Figure 3). Our findings indicated that reconstituted camel milk powder presented the highest foaming properties with 78.33 ± 2.63% and 480 ± 60 s for foam capacity (FC) and stability (FS),respectively.The skimmed camel milk (before drying) presented a FC and a FS of 68.33 ± 2.88% and 253 ± 50 s,respectively.

Furthermore, results of the current study indicated that the reconstituted bovine milk exhibited a FC and a FS of 72.33 ± 3.05% and 330 ± 36 s, respectively. These values were significantly higher than that observed for skimmed bovine milk (57.0 ± 2.63% and 210 ± 30 s for FC and FS, respectively). It is worth noticed that, either as skimmed or reconstituted, camel milk displayed higher FC and stability.

Similar findings were reported by [START_REF] Augustin | Skim milk powders with enhanced foaming and steamfrothing properties[END_REF]. These authors found that spray drying at 170 °C improved the FC of the bovine milk proteins. We admitted that the improvement of foam properties is highly depended on the physicochemical state of milk proteins. In fact, it was reported that during heat treatment, a partial deployment of whey proteins occurred [START_REF] Corredig | The mechanisms of the heat-induced interaction of whey proteins with casein micelles in milk[END_REF]. We hypothesize that, even if the time contact between the milk proteins and the warm-air was relatively short, a little whey proteins' denaturation could occur as a result of overdrying (low water content of the produced powders, Table 1). Such denaturation, as shown by SDS-PAGE and WPNI, enhanced the whey protein flexibility and participated in the improvement of the FC and stability [START_REF] Osorio | Effects of spray drying conditions and the addition of surfactants on the foaming properties of a whey protein concentrate[END_REF][START_REF] Schmidt | Effect of heat treatments on the functional properties of caseinate and whey protein isolate solutions[END_REF].

Food Engineering, Materials Science, & Nanotechnology

Camel milk powder characteristics…

CONCLUSION

In this work, three skimmed and one whole-fat camel milk powders were produced under various spray-drying parameters. The physical, biochemical, and rehydration characteristics, as well as the fat melting properties of these powders, were investigated and compared to those of bovine milk powders. Results of this study indicated that the composition of camel and bovine milks and powders was similar regarding the fat, protein, and lactose contents. However, the findings indicated that higher whey proteins and ash contents were observed in camel milk powders as compared to the bovine ones. In addition, the investigated camel and bovine milk powders presented a low d 50 (≤ 12μm) and a span ranging from 1.3 to 2.1. This indicated that these powders were composed of fine particles. Such kind of particles highly impacted the wettability and dispersibility of camel and bovine powders with no influence on their solubility (>99%). Furthermore, it is worth noticed the highest bulk density of camel milk powder as compared to those of bovine ones. Besides, analysis of the WPNI indicated that regardless of the fat content, camel milk powders (WPNI ≈ 9.6 g of N 2 /kg) were less denaturized than those of bovine ones (WPNI ≈ 8.5 g of N 2 /kg). This was linked to the absence of β-Lg from camel milk as indicated by the SDS-PAGE. The latter analysis together with the WPNI showed that camel and bovine milk proteins were relatively stable during spray drying (low denaturation extent). This trend was related to the low outlet drying temperature of 80 °C. This low denaturation extent improved the FC and stability of camel and bovine milk powders. The present work gives some insightful information about camel milk powder processing, which could help researchers and industrials to conduct further investigations.

  100 g of total powder fat. WPNI, whey protein nitrogen index. Same letter in the same row represents the statistical data significance (P>0.05).

Figure 1 -

 1 Figure 1-Differential scanning calorimetry thermograms recorded on heating of (A) whole-fat milk powders (a: camel; b: bovine) and (B) anhydrous fat extracted from the powders (a: camel; b: bovine).

Figure 2 -

 2 Figure 2-SDS-PAGE patterns of skimmed and reconstituted camel (A and B, respectively) and bovine (C and D, respectively) milks. Molecular Weight (MW): protein markers (10 to 100 kDa). Note: The proteins were identified based on the estimated molecular weights, the Uniprot Database (http://www.uniprot. org/), and the following studies: Ereifej et al. (2011); Felfoul et al. (2017); Omar et al. (2016).

Figure 3 -

 3 Figure 3-Foaming capacity and stability of skimmed (before drying) and reconstituted (before drying) camel and bovine milks. Same letter for each characteristic indicated the statistical data significance (P> 0.05).

Table 1 -Physicochemical composition of camel and bovine milks and their corresponding powders. Protein a w Total solids (%) Total protein (%)

 1

	Ash (%)
	Lactose (%)
	Fats (%)
	Whey proteins (%)
	Caseins (%)

%: g/100 g of milk or powder in dry basis.

Same letter in the same column for the samples category represents the statistical data significance (P>0.05).

Table 2 -Physicochemical characteristics of bovine and camel milk powders.

 2

					Skimmed milk powders			Whole-fat milk powder
	Inletࢨoutlet	160ࢨ80 °C	180ࢨ80 °C	200ࢨ80 °C	200ࢨ80	°C
	temperatures
	Milk powder	Bovine	Camel	Bovine	Camel	Bovine	Camel	Bovine	Camel
	Free fats	#	23.0 ± 0.1 a	10.0 ± 0.1 b	20.0 ± 0.1 c	10.0 ± 0.1 b	20.0 ± 0.1 c	15.0 ± 0.1 d	42.8 ± 1.0 e	25.4 ± 2.1 a
	WPNI (g of N 2 /kg)	8.59 ± 0.1 a	9.74 ± 0.1 b	8.58 ± 0.1 a	9.65 ± 0.1 b	8.51 ± 0.1 a	9.65 ± 0.1 b	8.63 ± 0.1 a	9.63 ± 0.1 b
	Size distribution
	d 50 (μm)	12.7 ± 0.1 a	12.6 ± 0.1 a	10.2 ± 0.1 b	9.2 ± 0.1 c	9.2 ± 0.1 c	8.8 ± 0.2 d	12.5 ± 0.1 a	10.5 ± 0.2 b
	Span		1

Journal of Food Science r Vol. 86, Iss. 1, 2021

Vol. 86, Iss. 1, 2021 r Journal of Food Science 105

Journal of Food Science r Vol. 86, Iss. 1, 2021

Vol. 86, Iss. 1, 2021 r Journal of Food Science 107

Journal of Food Science r Vol. 86, Iss. 1, 2021

Vol. 86, Iss. 1, 2021 r Journal of Food Science 109

Journal of Food Science r Vol. 86, Iss. 1, 2021

Vol. 86, Iss. 1, 2021 r Journal of Food Science 111

ACKNOWLEDGMENT

We would like to thank Mrs. Sawssen Derbel-Krichen for editing and improving the form of the manuscript.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

Ahmed Zouari was involved with the conceptualization, methodology, software, visualization, investigation, writingoriginal draft preparation, reviewing, and editing of the article.

Roua Lajnaf, Christelle Lopez, and Pierre Schuck contributed to the conceptualization, visualization, and investigation.

Hamadi Attia was responsible for supervision. Mohamed Ali Ayadi helped with the conceptualization, supervision, reviewing, and editing.