Long-term Antibody Production Research Articles

Potent immune responses against thermostable Foot-and-Mouth disease virus VP1 nanovaccine adjuvanted with polymeric thermostable scaffold

Foot-and-mouth disease (FMD) is an acute zoonosis causes significant economic losses. Vaccines able to stimulate efficient protective immune responses are urgently needed. In this study, Escherichia coli-derived recombinant VP1 of serotype A and O FMD virus (FMDV) was conjugated to thermostable scaffold lumazine synthase (LS) or Quasibacillus thermotolerans encapsulin (QtEnc) using a robust plug-and-display SpyTag/SpyCatcher system to generate multimeric nanovaccines. These nanovaccines induced highly potent antibody responses in vaccinated mice. On day 14 after the first immunisation, antibody titres were approximately 100 times higher than those of monomer antigens. Both vaccines induced high and long-term IgG antibody production. Moreover, the QtEnc-VP1 nanovaccine induced higher antibody titres than the LS-VP1 nanovaccine. The nanovaccines also induced Th1-biased immune responses and higher levels of neutralising antibodies. These data indicated that FMDV nanovaccines generated by conjugating VP1 with a thermostable scaffold are highly immunogenic and ideal candidates for FMDV control in low-resource areas.

Recombinant Hexon Protein as a New Bovine Adenovirus Type 3 Subunit Vaccine Candidate.

Bovine adenovirus (BAdV) type 3 causes respiratory and gastroenteric diseases of varying severity in cattle, particularly newborn calves. Trials have been conducted of a vaccination against the diseases caused by BAdV using both modified live-virus and inactivated-virus preparations in cattle, but no commercial BAdV-3 vaccine has yet reached the market. Therefore, there is an urgent need to develop new, safe, and effective vaccines against BAdV-3. Recombinant hexon protein (rhexon) of BAdV-3 was expressed in the E. coli system to evaluate immune response in mice and goats. Antibody responses and cytokine levels were analysed and the effects of administrations of different amounts of recombinant protein compared. Long-term antibody production was evaluated by indirect ELISA, and the total immunoglobulin G secreted by goats and mice immunised with the purified rhexon protein was determined. The immunised mice had a stronger antibody response than the control group at eight weeks post vaccination. The immunised groups also showed significantly higher (P ˂ 0.05) expression of interferon-γ, interleukin 2 (in mice), and interleukin 21 (in goats) at four weeks. Furthermore, vaccination with rhexon was able to induce long-term antibody production for at least 16 weeks in mice and goats. The rhexon protein induced immune responses, especially long-term antibody production and T helper 1 cell cytokine production in mice and goats. The immunogenic properties of this protein make it a promising subunit vaccine antigen.

Long-term antibody production and viremia in American mink (Neovison vison) challenged with Aleutian mink disease virus

BackgroundSelecting American mink (Neovison vison) for tolerance to Aleutian mink disease virus (AMDV) has gained popularity in recent years, but data on the outcomes of this activity are scant. The objectives of this study were to determine the long-term changes in viremia, seroconversion and survival in infected mink. Mink were inoculated intranasally with a local isolate of Aleutian mink disease virus (AMDV) over 4 years (n = 1742). The animals had been selected for tolerance to AMDV for more than 20 years (TG100) or were from herds free of AMDV (TG0). The progenies of TG100 and TG0, and their crosses with 25, 50 and 75% tolerance ancestry were also used. Blood samples were collected from each mink up to 14 times until 1211 days post-inoculation (dpi) and were tested for viremia by PCR and for anti-AMDV antibodies by counter-immunoelectrophoresis (CIEP). Viremia and CIEP status were not considered when selecting replacements. Low-performing animals were pelted and the presence of antibodies in their blood and antibody titer were measured by CIEP, and viremia and viral DNA in seven organs (n = 936) were tested by PCR.ResultsThe peak incidences of viremia (66.7%) and seropositivity (93.5%) were at 35 dpi. The incidence of viremia decreased over time while the incidence of seroconversion increased. The least-squares means of the incidence of PCR positive of lymph node (0.743) and spleen (0.656) were significantly greater than those of bone marrow, liver, kidneys, lungs and small intestine (0.194 to 0.342). Differences in tolerant ancestry were significant for every trait measured. Incidences of viremia over time, terminal viremia, seropositivity over time, AMDV DNA in organs and antibody titer were highest in the susceptible groups (TG0 or TG25) and lowest in the tolerant groups (TG100 or TG75).ConclusionPrevious history of selection for tolerance resulted in mink with reduced viral replication and antibody titer. Viremia had a negative effect and antibody production had a positive effect on survival and productivity.

Proteasomal adaptations underlying carfilzomib-resistance in human bone marrow plasma cells.

Donor-specific antibodies (DSAs) have a deleterious effect on allografts and remain a major immunologic barrier in transplantation. Current therapies to eliminate DSAs are ineffective in highly HLA-sensitized patients. Proteasome inhibitors have been employed as a strategy to target bone marrow plasma cells (BMPCs), the source of long-term antibody production; however, their efficacy has been limited by poorly defined drug-resistance mechanisms. Here, we performed transcriptomic profiling of CD138+ BMPCs that survived in vivo desensitization therapy with the proteasome inhibitor carfilzomib to identify mechanisms of drug resistance. The results revealed a genomic signature that included increased expression of the immunoproteasome, a highly specialized proteasomal variant. Western blotting and functional studies demonstrated that catalytically active immunoproteasomes and the immunoproteasome activator PA28 were upregulated in carfilzomib-resistant BMPCs. Carfilzomib-resistant BMPCs displayed reduced sensitivity to the proteasome inhibitors carfilzomib, bortezomib, and ixazomib, but enhanced sensitivity to an immunoproteasome-specific inhibitor ONX-0914. Finally, in vitro carfilzomib treatment of BMPCs from HLA-sensitized patients increased levels of the immunoproteasome β5i (PSMB8) catalytic subunit suggesting that carfilzomib therapy directly induces an adaptive immunoproteasome response. Taken together, our results indicate that carfilzomib induces structural changes in proteasomes and immunoproteasome formation.

Vaccine Designs Utilizing Invariant NKT-Licensed Antigen-Presenting Cells Provide NKT or T Cell Help for B Cell Responses.

Vaccines against a variety of infectious diseases have been developed and tested. Although there have been some notable successes, most are less than optimal or have failed outright. There has been discussion about whether either B cells or dendritic cells (DCs) could be useful for the development of antimicrobial vaccines with the production of high titers of antibodies. Invariant (i)NKT cells have direct antimicrobial effects as well as adjuvant activity, and iNKT-stimulated antigen-presenting cells (APCs) can determine the form of the ensuing humoral and cellular immune responses. In fact, upon activation by ligand, iNKT cells can stimulate both B cells and DCs as via either cognate or non-cognate help. iNKT-licensed DCs generate antigen-specific follicular helper CD4+ T cells, which in turn stimulate B cells, thus leading to long-term antigen-specific antibody production. Follicular helper iNKT cell-licensed B cells generally produce rapid, but short-term antibody. However, under some conditions in the presence of Th cells, the antibody production can be prolonged. With regards to humoral immunity, the quality and quantity of Ab produced depends on the APC type and the form of the vaccine. In terms of cellular immunity and, in particular, the induction of cytotoxic CD8+ T cells, iNKT-licensed DCs show prominent activity. In this review, we discuss differences in iNKT-stimulated APC types and the quality of the ensuing immune response, and also discuss their application in vaccine models to develop successful preventive immunotherapy against infectious diseases.

CD4 T cell autophagy is integral to memory maintenance

Studies of mice deficient for autophagy in T cells since thymic development, concluded that autophagy is integral to mature T cell homeostasis. Basal survival and functional impairments in vivo, limited the use of these models to delineate the role of autophagy during the immune response. We generated Atg5f/f distal Lck (dLck)-cre mice, with deletion of autophagy only at a mature stage. In this model, autophagy deficiency impacts CD8+ T cell survival but has no influence on CD4+ T cell number and short-term activation. Moreover, autophagy in T cells is dispensable during early humoral response but critical for long-term antibody production. Autophagy in CD4+ T cells is required to transfer humoral memory as shown by injection of antigen-experienced cells in naive mice. We also observed a selection of autophagy-competent cells in the CD4+ T cell memory compartment. We performed in vitro differentiation of memory CD4+ T cells, to better characterize autophagy-deficient memory cells. We identified mitochondrial and lipid load defects in differentiated memory CD4+ T cells, together with a compromised survival, without any collapse of energy production. We then propose that memory CD4+ T cells rely on autophagy for their survival to regulate toxic effects of mitochondrial activity and lipid overload.

ATP-degrading ENPP1 is required for survival (or persistence) of long-lived plasma cells

Survival of antibody-secreting plasma cells (PCs) is vital for sustained antibody production. However, it remains poorly understood how long-lived PCs (LLPCs) are generated and maintained. Here we report that ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is preferentially upregulated in bone marrow LLPCs compared with their splenic short-lived counterparts (SLPCs). We studied ENPP1-deficient mice (Enpp1−/−) to determine how the enzyme affects PC biology. Although Enpp1−/− mice generated normal levels of germinal center B cells and plasmablasts in periphery, they produced significantly reduced numbers of LLPCs following immunization with T-dependent antigens or infection with plasmodium C. chabaudi. Bone marrow chimeric mice showed B cell intrinsic effect of ENPP1 selectively on generation of bone marrow as well as splenic LLPCs. Moreover, Enpp1−/− PCs took up less glucose and had lower levels of glycolysis than those of wild-type controls. Thus, ENPP1 deficiency confers an energetic disadvantage to PCs for long-term survival and antibody production.

Plasma cell survival in the absence of B cell memory

Pre-existing serum antibodies play an important role in vaccine-mediated protection against infection but the underlying mechanisms of immune memory are unclear. Clinical studies indicate that antigen-specific antibody responses can be maintained for many years, leading to theories that reactivation/differentiation of memory B cells into plasma cells is required to sustain long-term antibody production. Here, we present a decade-long study in which we demonstrate site-specific survival of bone marrow-derived plasma cells and durable antibody responses to multiple virus and vaccine antigens in rhesus macaques for years after sustained memory B cell depletion. Moreover, BrdU+ cells with plasma cell morphology can be detected for 10 years after vaccination/BrdU administration, indicating that plasma cells may persist for a prolonged period of time in the absence of cell division. On the basis of these results, long-lived plasma cells represent a key cell population responsible for long-term antibody production and serological memory.

In vivo dendritic cell targeting cellular vaccine induces CD4+ Tfh cell-dependent antibody against influenza virus.

An induction of long-term cellular and humoral immunity is for the goal of vaccines, but the combination of antigens and adjuvant remain unclear. Here, we show, using a cellular vaccine carrying foreign protein antigen plus iNKT cell glycolipid antigen, designated as artificial adjuvant vector cells (aAVCs), that mature XCR1− DCs in situ elicit not only ordinal antigen-specific CD4+T cells, but also CD4+ Tfh and germinal center, resulted in inducing long-term antibody production. As a mechanism for leading the long-term antibody production by aAVC, memory CD4+ Tfh cells but not iNKTfh cells played an important role in a Bcl6 dependent manner. To develop it for influenza infection, we established influenza hemagglutinin-carrying aAVC (aAVC-HA) and found that all the mice vaccinated with aAVC-HA were protected from life-threatening influenza infection. Thus, the in vivo DC targeting therapy by aAVC would be useful for protection against viral infection.

CD28 Induces Mitochondrial Respiration through Slp-76 in Long-Lived Plasma Cells for Reactive Oxygen Species-Dependent Survival

Sustained humoral immunity is dependent upon the production of neutralizing antigen-specific antibodies by plasma cells. Upon antigen activation, B cells differentiate into short-lived plasma cells (SLPCs) which home to secondary lymphoid organs such as the spleen where they live for days to weeks before dying by apoptosis. However, there are examples in the human population in which neutralizing antibody titers persist despite a lack of continual antigen exposure. Therefore an expanded model has been proposed wherein activated B cells differentiate into long-lived plasma cells (LLPCs) that home to the bone marrow and survive indefinitely in specialized survival niches. They are not intrinsically long-lived, but rather depend upon extrinsic survival signals for their persistence and long-term antibody production. Furthermore, many of these survival signals are shared with their malignant counterpart in Multiple Myeloma (MM). Our work centers on the cellular and molecular mechanisms by which MM and LLPCs survive in the bone marrow microenvironment.Our lab has recently demonstrated that CD28 provides an intrinsic survival signal to LLPCs. Plasma cells deficient in CD28 or mutated in the CD28-Grb2/Vav binding region don't survive and cannot sustain antigen-specific antibody titers post-vaccination. CD28 also provides a survival signal in MM under chemotherapeutic stress. CD28 is best characterized as the canonical co-stimulatory molecule in T cells governing metabolic fitness. Plasma cells and MM are highly biosynthetic and require metabolic expenditure. However, the molecular and metabolic pathways that provide this CD28-mediated survival advantage in LLPCs/MM are not well understood.Here we demonstrate that CD28 enhances mitochondrial membrane potential in LLPCs and MM, but not in SLPCs. In addition, CD28 induces mitochondrial biogenesis in LLPC and MM. By using oxygen consumption as a direct readout of mitochondrial respiration, we show that CD28 activation increases oxidative phosphorylation. A major byproduct of respiration is the production of reactive oxygen species (ROS). CD28 activation induces mitochondrial-derived ROS in LLPCs but not SLPCs, as seen using the mitoROS specific dye MitoSox. Furthermore, in the mutant LLPCs that cannot signal through CD28-Grb2/Vav, CD28 cannot induce ROS.ROS are well-characterized cell-damaging agents. In order to elucidate the effects of ROS on LLPCs, we specifically inhibited ROS produced by the mitochondria with MnTBAP. Paradoxically, inhibiting ROS prevents CD28-mediated survival. Taken together the data suggest that CD28 increases mitochondrial respiration for ROS-dependent survival. By analyzing open-source RNAseq data acquired from purified populations of bone marrow LLPCs and splenic SLPCs, the LLPC population exhibits higher transcript levels of genes associated with mitochondrial respiration, oxidative phosphorylation, and the TCA cycle. However, the signals that drive this metabolic program are not characterized.Building upon our previous findings that the CD28 Grb2/Vav binding moiety on the cytoplasmic tail is required for CD28-mediated survival, we interrogated this signaling axis. Upon CD28 activation, Vav colocalizes to the CD28 receptor seen via ImageStream technology. A major adaptor protein downstream of Vav, Slp-76, is highly expressed in LLPCs and MM but not in SLPCs at the protein level as evidenced by intracellular flow cytometry. By using a transcriptomic approach, Slp-76 is also highly upregulated in human LLPCs compared to SLPCs. We then examined the ability of CD28 to provide a prosurvival signal via genetic approaches wherein Slp-76 was knocked-out or mutated at the Vav binding region. In either case, CD28 was not capable of inducing survival to LLPCs. When examining the knock-in mice that cannot signal through Vav/Slp-76, the LLPC population with high levels of ROS is completely absent when compared to wild type LLPCs. Interestingly, CD28 upregulates Slp-76 at mRNA and protein level in both SLPC and LLPC, suggesting that CD28 may be capable of determining plasma cell fate at the onset of differentiation.Taken together the data suggests that CD28 may regulate LLPC induction, survival and function through Grb2/Vav/Slp-76 mediated metabolic reprogramming. This makes CD28 an attractive target in vaccine design and for treatment of both autoimmune disorders and multiple myeloma. DisclosuresNo relevant conflicts of interest to declare.

Quantitative image analysis of cell colocalization in murine bone marrow

Long-term antibody production is a key property of humoral immunity and is accomplished by long-lived plasma cells. They mainly reside in the bone marrow, whose importance as an organ hosting immunological memory is becoming increasingly evident. Signals provided by stromal cells and eosinophils may play an important role for plasma cell maintenance, constituting a survival microenvironment. In this joint study of experiment and theory, we investigated the spatial colocalization of plasma cells, eosinophils and B cells by applying an image-based systems biology approach. To this end, we generated confocal fluorescence microscopy images of histological sections from murine bone marrow that were subsequently analyzed in an automated fashion. This quantitative analysis was combined with computer simulations of the experimental system for hypothesis testing. In particular, we tested the observed spatial colocalization of cells in the bone marrow against the hypothesis that cells are found within available areas at positions that were drawn from a uniform random number distribution. We find that B cells and plasma cells highly colocalize with stromal cells, to an extent larger than in the simulated random situation. While B cells are preferentially in contact with each other, i.e., form clusters among themselves, plasma cells seem to be solitary or organized in aggregates, i.e., loosely defined groups of cells that are not necessarily in direct contact. Our data suggest that the plasma cell bone marrow survival niche facilitates colocalization of plasma cells with stromal cells and eosinophils, respectively, promoting plasma cell longevity.

Adjuvant-specific regulation of long-term antibody responses by ZBTB20

The duration of antibody production by long-lived plasma cells varies with the type of immunization, but the basis for these differences is unknown. We demonstrate that plasma cells formed in response to the same immunogen engage distinct survival programs depending on the adjuvant. After alum-adjuvanted immunization, antigen-specific bone marrow plasma cells deficient in the transcription factor ZBTB20 failed to accumulate over time, leading to a progressive loss of antibody production relative to wild-type controls. Fetal liver reconstitution experiments demonstrated that the requirement for ZBTB20 was B cell intrinsic. No defects were observed in germinal center numbers, affinity maturation, or plasma cell formation or proliferation in ZBTB20-deficient chimeras. However, ZBTB20-deficient plasma cells expressed reduced levels of MCL1 relative to wild-type controls, and transgenic expression of BCL2 increased serum antibody titers. These data indicate a role for ZBTB20 in promoting survival in plasma cells. Strikingly, adjuvants that activate TLR2 and TLR4 restored long-term antibody production in ZBTB20-deficient chimeras through the induction of compensatory survival programs in plasma cells. Thus, distinct lifespans are imprinted in plasma cells as they are formed, depending on the primary activation conditions. The durability of vaccines may accordingly be improved through the selection of appropriate adjuvants.

ZBTB20 is an adjuvant-specific regulator of long-term antibody responses (P1439)

Abstract The duration of antibody production following immunization can vary greatly depending on the vaccine, but the molecular basis for these differences is unknown. Here, we demonstrated that ZBTB20 is an adjuvant-specific transcription factor critical for the durable antibody production by long-lived plasma cells. ZBTB20 was highly expressed in germinal center and memory B cells and in short and long-lived plasma cells, but not in naïve B cells.Zbtb20 deficiency due to a gene trap mutation in the Zbtb20 locus specifically led to defective accumulation of long-lived plasma cells following immunization, while short-lived plasmablasts and germinal center B cells were unaffected. Competitive fetal liver reconstitution experiments demonstrated a B cell-intrinsic requirement for ZBTB20 in long-term antibody production following alum-adjuvanted immunization. Strikingly, inclusion of Toll-like receptor ligands in the adjuvant completely rescued long-term antibody production in Zbtb20-deficient fetal liver chimeras. Thus, different immunization conditions induce long-lived plasma cells with qualitatively distinct transcriptional programs to regulate their turn-over.

Mechanisms that determine plasma cell lifespan and the duration of humoral immunity

Summary: Humoral immunity following vaccination or infection is mainly derived from two types of cells: memory B cells and plasma cells. Memory B cells do not actively secrete antibody but instead maintain their immunoglobulin in the membrane‐bound form that serves as the antigen‐specific B‐cell receptor. In contrast, plasma cells are terminally differentiated cells that no longer express surface‐bound immunoglobulin but continuously secrete antibody without requiring further antigenic stimulation. Pre‐existing serum or mucosal antibody elicited by plasma cells (or other intermediate antibody‐secreting cells) represents the first line of defense against reinfection and is critical for protection against many microbial diseases. However, the mechanisms involved with maintaining long‐term antibody production are not fully understood. Here, we examine several models of long‐term humoral immunity and present a new model, described as the ‘Imprinted Lifespan’ model of plasma cell longevity. The foundation of this model is that plasma cells are imprinted with a predetermined lifespan based on the magnitude of B‐cell signaling that occurs during the induction of an antigen‐specific humoral immune response. This represents a testable hypothesis and may explain why some antigen‐specific antibody responses fade over time whereas others are maintained essentially for life.

Guidance of B Cells by the Orphan G Protein-Coupled Receptor EBI2 Shapes Humoral Immune Responses

Humoral immunity depends on both rapid and long-term antibody production against invading pathogens. This is achieved by the generation of spatially distinct extrafollicular plasmablast and follicular germinal center (GC) B cell populations, but the signals that guide responding B cells to these alternative compartments have not been fully elucidated. Here, we show that expression of the orphan G protein-coupled receptor Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) by activated B cells was essential for their movement to extrafollicular sites and induction of early plasmablast responses. Conversely, downregulation of EBI2 enabled B cells to access the center of follicles and promoted efficient GC formation. EBI2 therefore provides a previously uncharacterized dimension to B cell migration that is crucial for coordinating rapid versus long-term antibody responses.

Three‐dimensional culture for monoclonal antibody production by hybridoma cells immobilized in macroporous gel particles

Cell proliferation and long-term production of monoclonal antibody IgG(2b) by M2139 hybridoma cells immobilized in macroporous gel particles (MGPs) in packed-bed reactor were studied for a period of 60 days. The MGPs were made of supermacroporous gels produced in frozen conditions from crosslinked polyacrylamide and modified with gelatin which were housed in special plastic carriers (7 x 9 mm(2)). Cells were trapped in the interior part of MGPs by attaching to the void space of the gel matrix as three-dimensional (3D) cultivation using gelatin as a substrate layer. Optimizing productivity by hybridoma cell relies on understanding regulation of antibody production. In this study, the behavior of M2139 cells in two-dimensional cultures on multiwell plate surfaces was also investigated. The effect of three different medium such as basal medium Dulbecco's modified Eagle's medium (D-MEM) containing L-glutamine or L-glutamine + 2 mM alpha-ketoglutarate or L-alanyl-glutamine (GlutaMAXtrade mark) was studied prior to its use in 3D cultivation. The kinetics of cell growth in basal medium containing L-glutamine + alpha-ketoglutarate was similar to cells grown on GlutaMAX containing medium, whereas D-MEM containing L-glutamine showed lower productivity. With the maximal viable cell density (6.85 x 10(6) cells mL(-1)) and highest specific mAb production rate (3.9 mug mL(-1) 10(-4) viable cell day(-1)), D-MEM-GlutaMAX was further selected for 3D cultivation. Cells in MGPs were able to grow and secrete antibody for 30 days in packed-bed batch reactor, before a fresh medium reservoir was replaced. After being supplied with fresh medium, cells again showed continuous growth for another 30 days with mAb production efficiency of 50%. These results demonstrate that MGPs can be used efficiently as supporting carrier for long-term monoclonal antibody production.

Application of a Cell-Once-Through Perfusion Strategy for Production of Recombinant Antibody from rCHO Cells in a Centritech Lab II Centrifuge System

Based upon the results of scale-down intermittent perfusion processes, a cell-once-through (COT) perfusion concept was applied to a dual bioreactor system coupled to a Centritech Lab II centrifuge for culture of recombinant Chinese hamster ovary (rCHO) cells for monoclonal antibody production. In this new culture mode, i.e., the COT perfusion process, total spent medium was transferred to the centrifuge and a fixed percentage was removed. Approximately 99% of the viable cells are transferred to another bioreactor filled with fresh medium by single operation of the Centritech Lab II centrifuge system for about 30 min. Accordingly, a significant reduction of the cell-passage frequency to the centrifuge led to minimization of cell damage caused by mechanical shear stress, oxygen limitation, nutrient limitation, and low temperature outside the bioreactor. The effects of culture temperature shift and fortified medium on cell growth and recombinant antibody production in the COT perfusion process were investigated. Although the suppressive effects of low culture temperature on cell growth led to a loss of stability in a long-term COT perfusion culture system, the average antibody concentration at 33 degrees C was 157.8 mg/L, approximately 2.4-fold higher than that at 37 degrees C. By the use of a fortified medium at 37 degrees C, rCHO cells were maintained at high density above 1.2 x 10(7) cells/mL, and antibody was produced continuously in a range of 260-280 mg/L in a stable long-term COT perfusion culture. The proposed new culture mode, the COT perfusion approach, guarantees the recovery of rCHO cells damaged by lowered temperature or high lactate and ammonium concentration. It will be an attractive choice for minimization of cell damage and stable long-term antibody production with high cell density.

Plasma cell S1P1 expression determines secondary lymphoid organ retention versus bone marrow tropism

After induction in secondary lymphoid organs, a subset of antibody-secreting cells (ASCs) homes to the bone marrow (BM) and contributes to long-term antibody production. The factors determining secondary lymphoid organ residence versus BM tropism have been unclear. Here we demonstrate that in mice treated with FTY720 or that lack sphingosine-1-phosphate (S1P) receptor-1 (S1P1) in B cells, IgG ASCs are induced and localize normally in secondary lymphoid organs but they are reduced in numbers in blood and BM. Many IgG ASCs home to BM on day 3 of the secondary response and day 3 splenic ASCs exhibit S1P responsiveness, whereas the cells remaining at day 5 are unable to respond. S1P1 mRNA abundance is higher in ASCs isolated from blood compared to spleen, whereas CXCR4 expression is lower. Blood ASCs also express higher amounts of Kruppel-like factor (KLF)2, a regulator of S1P1 gene expression. These findings establish an essential role for S1P1 in IgG plasma cell homing and they suggest that differential regulation of S1P1 expression in differentiating plasma cells may determine whether they remain in secondary lymphoid organs or home to BM.

Prevention of long-term IgE antibody production by gene gun–mediated DNA vaccination

Vaccination with allergen-encoding DNA represents a promising approach for the treatment of allergic diseases. In a mouse model of type I allergy, we analyzed the ability of biolistic transfection to inhibit antigen-specific IgE production and to modulate TH2 responses. BALB/c mice were vaccinated by means of gene gun-mediated DNA immunization with plasmid vector pCMV-betaGal, encoding beta-galactosidase as a model allergen. Subsequently, mice were immunized by means of repeated intraperitoneal injection of beta-galactosidase adsorbed to the adjuvant aluminum hydroxide. Development of IgE, IgG1, and IgG2a antibody titers during the course of immunization was followed, and anaphylactic potential of sera was determined by using RBL-2H3 degranulation assay. Spleen cells of vaccinated mice and unvaccinated control animals were stimulated in vitro to analyze cytokine production and induction of CD8 + effector T cells. Gene gun-mediated DNA immunization with pCMV-betaGal very efficiently prevented IgE antibody production on a long-term basis. Concomitantly, IgG1 antibody levels in vaccinated mice were strongly reduced, whereas IgG2a antibody production was increased. Analysis of cytokine profiles indicated immune deviation from a TH2-biased response in control mice toward a mixed TH1/TH2 response in vaccinated mice. In addition, substantial numbers of IFN-gamma-producing CD8 + effector T cells were found in vaccinated mice. Gene gun-mediated DNA vaccination prevents the induction of long-lasting IgE antibody production.

A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice.

The recent emergence of severe acute respiratory syndrome (SARS) was caused by a novel coronavirus, SARS-CoV. It spread rapidly to many countries and developing a SARS vaccine is now urgently required. In order to study the immunogenicity of UV-inactivated purified SARS-CoV virion as a vaccine candidate, we subcutaneously immunized mice with UV-inactivated SARS-CoV with or without an adjuvant. We chose aluminum hydroxide gel (alum) as an adjuvant, because of its long safety history for human use. We observed that the UV-inactivated SARS-CoV virion elicited a high level of humoral immunity, resulting in the generation of long-term antibody secreting and memory B cells. With the addition of alum to the vaccine formula, serum IgG production was augmented and reached a level similar to that found in hyper-immunized mice, though it was still insufficient to elicit serum IgA antibodies. Notably, the SARS-CoV virion itself was able to induce long-term antibody production even without an adjuvant. Anti-SARS-CoV antibodies elicited in mice recognized both the spike and nucleocapsid proteins of the virus and were able to neutralize the virus. Furthermore, the UV-inactivated virion induced regional lymph node T-cell proliferation and significant levels of cytokine production (IL-2, IL-4, IL-5, IFN-γ and TNF-α) upon restimulation with inactivated SARS-CoV virion in vitro. Thus, a whole killed virion could serve as a candidate antigen for a SARS vaccine to elicit both humoral and cellular immunity.