Production Of Human Antibodies Research Articles

Research Highlights

Research Highlights

Teaching baby B cells

Adaptive Immunity Antibodies are central to immune defense. B cells can generate several antibody isotypes that fulfill different functional roles, and repeated antigen exposure induces antibody somatic hypermutation. Nielsen et al. studied a longitudinal birth cohort to determine how antigen exposure early in life affects human antibody production. Somatic hypermutation increased as the children got older, and certain VH genes were associated with different isotypes. Increased pathogen exposure was associated with immunoglobulin D (IgD) and IgM mutations. Interestingly, children with eczema or allergies had higher rates of IgE mutation. These valuable data shed light on how human B cells grow up. Sci. Transl. Med. 11 , eaat2004 (2019).

206 Towards the generation of transchromosomic goats for the production of fully human immunoglobulin

We have previously reported that inactivation of endogenous immunoglobulin (Ig) genes can enhance the production of human polyclonal antibodies (hpAb) in transchromosomic (Tc) cattle containing a human artificial chromosome (HAC) comprising the entire human Ig gene repertoire. Goats offer the advantages of having a shorter gestation period and growing to adult size much faster than larger ungulates. Therefore, based upon our previously established Tc cattle platform, in this study, we aimed to generate immunoglobulin lambda light chain knockout (IGLλ −/− )/HAC Tc goats expressing hpAb using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT) techniques. First, we designed multiple specific single-guide RNAs (sgRNA) targeting the coding sequences of constant regions of goat IGLλ1, IGLλ2 (both in NC_030824.1), and IGLλ3 (NW_017189530.1) and constructed corresponding gene targeting vectors. Gene targeting efficiency analysis in goat fibroblasts showed that the sgRNA were efficient in directing Cas9 to generate targeted indels in corresponding IGLλ genes with efficiencies ranging between 20 and 30%. We then targeted the IGLλ genes and generated a cloned IGLλ −/− fetus by SCNT (first round of cloning; G1) using single cell-derived IGLλ −/− fibroblast colonies as nuclear donors and established IGLλ −/− fetal fibroblast cell lines. Second, we established IGLλ −/− /HAC fibroblast colonies by transferring the isKcHACΔ, a HAC containing the entire unrearranged human immunoglobulin heavy-chain and kappa-chain sequences, into the IGLλ −/− fetal fibroblast cells via microcell-mediated chromosome transfer (MMCT; Matsushita et al. 2014 PLoS One 9, e90383). The resulting IGLλ −/− /HAC fibroblasts were used as nuclear donors for the production of IGLλ −/− /HAC goats by SCNT (second round of cloning; G2). A total of 311 one-cell stage G2 embryos were generated using 4 IGLλ −/− /HAC fetal fibroblast colonies and surgically transferred into 19 oestrus-synchronized recipients. Seven pregnancies were confirmed (7/19; 36.8%) with ultrasonography at Day 60±3 of gestation, and 1 pregnancy (1/7; 14.3%) developed to term, leading to the birth of a live and healthy kid. Genetic and serological characterisation of this Tc goat produced by the second round of SCNT is in progress.

Increased CD40L+PD-1+ follicular helper T cells (Tfh) as a biomarker for predicting calcineurin inhibitor sensitivity against Tfh-mediated B-cell activation/antibody production after kidney transplantation.

It is unclear to what extent the development of follicular helper T cells (Tfh) and de novo donor-specific human leukocyte antigen antibody (DSA) production could be influenced by immunosuppressive agents, particularly calcineurin inhibitor (CNI; cyclosporine or tacrolimus), after kidney transplantation. Here, the effects of immunosuppressive agents on Tfh-mediated B-cell activation and antibody production were investigated. In vitro circulating Tfh (cTfh; memory CD4+CXCR5+)/B-cell (CD19+) co-culture assays revealed that CNI considerably inhibited cTfh-mediated B-cell activation and IgG antibody secretion through the suppression of IL-21 and IL-2. Both IL-21 and CD40L up-regulated IL-2 receptors (CD25) on B cells, and anti-CD25 antibody induced apoptosis of activated B cells, resulting in the inhibition of IgG production. The frequency of cTfh-expressed CD40L and PD-1 was elevated in patients with de novo DSA 1 year after transplantation. The degree of inhibition by CNI was dependent on Staphylococcal enterotoxin B-induced CD40L+PD-1+ cTfh up-regulation level. Our data demonstrate that CD40L+PD-1+cTfh could be a marker to implicate individual difference in CNI sensitivity for Tfh-mediated B-cell activation in kidney transplantation.

Significance of humanized mouse models for evaluating humoral immune response against cancer vaccines

Abstract Because accumulation of mutations produces unique neoantigen patterns in cancer cells, it has been hypothesized that these patterns are recognized by the patients' immune system. Therefore, the development of cancer vaccines has been challenging owing to the possibility of an anti-cancer effect induced by the immune system against such neoantigens. However, it is difficult to develop effective vaccines because of the variety of mutations induced in cancer cells and human leukocyte antigen (HLA), which is predicted to present the neoantigen-derived peptides. Moreover, the activation of cancer-specific cytotoxic cells is inhibited by cancer cell immunoediting in each patient. Although cellular immunity can be analyzed ex vivo, there are no definite methods to evaluate humoral immunity. A humanized mouse model has been developed and used for evaluating the multipotency of hematopoietic stem cells. Presently, significant improvements in the humanized mouse model approach have partially recapitulated human humoral immunity in vivo, and human antibody production can be induced in the mouse model. These mice can be used to produce new cancer vaccines and to establish polypharmacy protocols in a preclinical model. In this review, we discuss the preclinical evaluation of the cancer vaccines using humanized mice transferred with patient-derived peripheral blood mononuclear cells (PBMCs) to advance a personalized medicine approach.

Glycoengineering design options for IgG1 in CHO cells using precise gene editing

Precise gene editing technologies are providing new opportunities to stably engineer host cells for recombinant production of therapeutic glycoproteins with different glycan structures. The glycosylation of recombinant therapeutics has long been a focus for both quality and consistency of products and for optimizing and improving pharmacokinetic properties as well as bioactivity. Structures of glycans on therapeutic glycoproteins are important for circulation, biodistribution and bioactivity. In particular, the latter has been demonstrated for therapeutic IgG1 antibodies where the core α1,6Fucose on the conserved N-glycan at Asn297 have remarkable dampening effects on antibody effector functions. We previously explored precise gene engineering and design options for N-glycosylation in CHO cells, and here we focus on engineering options possible for N-glycans on human IgG1. We demonstrate stable precise gene engineering of rather homogenous biantennary N-glycans with and without galactose (G0F, G2F) as well as the α2,6-linked monosialylated (G2FS1) glycoform. We were unable to introduce substantial disialylated glycoforms. Instead we engineered a novel monoantennary homogeneous N-glycan design with complete α2,6-linked sialic acid capping. All N-glycoforms may be engineered with and without core α1,6Fucose. The stably engineered design options enable production of human IgG antibodies with an array of distinct glycoforms for testing and selection of optimal design for different therapeutic applications.

Evaluating Human Immune Responses for Vaccine Development in a Novel Human Spleen Cell-Engrafted NOD-SCID-IL2rγNull Mouse Model.

The lack of preclinical models able to faithfully predict the immune responses which are later obtained in the clinic is a major hurdle for vaccines development as it increases markedly the delays and the costs required to perform clinical studies. We developed and evaluated the relevance to human immune responses of a novel humanized mouse model, humanized-spleen cells-NOD-SCID-gamma null (Hu-SPL-NSG), in which we grafted human spleen cells in immunodeficient NOD-SCID-IL-2rγnull (NSG) mice. We selected the malaria vaccine candidate, Liver Stage Antigen 3-Full Length, because we had previously observed a major discrepancy between preclinical and clinical results, and compared its immunogenicity with that of a shorter form of the molecule, LSA3-729. NSG mice engrafted with human spleen lymphocytes were immunized with either LSA3-FL or LSA3-729, both adjuvanted with montanide ISA720. We found that the shorter LSA3-729 triggered the production of human antibodies and a T-helper-type 1 cellular immune response associated with protection whereas LSA3-FL did not. Results were consistent in five groups receiving lymphocytes from five distinct human donors. We identified antigenic regions in the full-length molecule, but not in the shorter version, which induced T-regulatory type of cellular responses. These regions had failed to be predicted by previous preclinical experiments in a wide range of animal models, including primates. Results were reproducible using spleen cells from all five human donors. The findings in the Hu-SPL-NSG model were similar to the results obtained using LSA3-FL in the clinic and hence could have been used to predict them. The model does not present graft versus host reaction, low survival of engrafted B lymphocytes and difficulty to raise primary immune responses, all limitations previously reported in humanized immune-compromised mice. Results also point to the shorter construct, LSA3-729 as a more efficient vaccine candidate. In summary, our findings indicate that the Hu-SPL-NSG model could be a relevant and cost-saving choice for early selection of vaccine candidates before clinical development, and deserves being further evaluated.

Production of human single-chain fragment antibody (ScFv) against human epidermal growth factor receptor-2 (HER-2) by phage display technology.

Breast cancer with more than 1.7 million diagnoses per year has been known as one of the most prevalent cancers among women worldwide. Despite the availability of advanced treatment options, cancer progression and metastasis is observed in 20% of patients. Human epidermal growth factor receptor-2 (HER-2) is considered as an important prognostic and diagnostic tumor marker for breast cancer. While HER-2 is expressed on the surface of normal cells, its overexpression occurs in 20-25% on breast cancer tumor cells. This type of tumor which is referred to as HER-2+ is the most aggressive type of breast cancer and shows more resistance to radiotherapy. Single-chain fragment antibodies (ScFvs) offer several advantages in comparison to conventional whole antibodies due to their small size. Particularly, ScFv fragments show improved diffusion and solid tumor penetration. In this study, a human ScFv antibody library was used to isolate anti-HER-2 ScFv antibodies through cell panning and mix antigen-cell panning strategies. Analysis of the binding activity and specificity of isolated ScFv antibodies against HER-2-expressing cell lines and recombinant HER-2 antigen indicated the higher efficiency of the cell panning strategy in isolation of ScFv antibody fragments.

Construction of a human recombinant polyclonal Fab fragment antibody library using peripheral blood lymphocytes of snake bitten victims

Human snake bitten poisoning is a serious threat in many tropical and subtropical countries such as Iran. The best acceptable treatment of envenomated humans is antivenoms; however they have a series of economic and medical problems and need more improvements. In this study a combinatorial human immunoglobulin gene library against some of Iranian snakes venoms was constructed. Total RNA prepared from peripheral blood lymphocytes of two recovered snake victims. RT-PCR was used for cDNA synthesis and amplification of the heavy (Fd segment) and kappa light chains of IgG antibody. After digestion of the heavy chain with SpeI and XhoI and light chain with XbaI and SacI enzymes, inserted successively into the cloning vector pComb3x, and then recombinant vector transformed to TG1 bacteria to construct the Fab library. For determination insertion rate of Fab segment into cloning vector, plasmids of 12 clones of library were extracted and digested with SfiI. Length of amplified Fd and κ chains, were 650 - 750 bp. Primary library size was determined to contain 4.9×105 members out of which half of them contained the same size of Fab fragment. This result is comparable to some researchers and shows that this method could be appropriate tool for the production of human polyclonal Fab fragment antibodies for management of poisonous snake bitted victims.

Therapeutic potential of regulatory cytokines that target B cells.

Autoreactive B cells play a crucial role in the pathogenesis of autoimmune diseases by producing auto-antibodies and presenting antigens. Regulatory cytokines that simultaneously suppress multiple pathways have the potential to control autoreactive B cells. The generally inhibitory cytokine IL-10 may have a stimulatory effect on human B-cell survival and antibody production. TGF-β family cytokines can decrease or increase antibody production and can suppress B-cell proliferation and differentiation. In contrast to TGF-β1, which induces extensive fibrosis, TGF-β3 and bone morphogenetic protein 6 (BMP-6)/BMP-7 induce non-scarring wound healing and counteract tissue fibrosis. Therefore, TGF-β3 and BMP-6/BMP-7 may be clinically applicable as therapeutic cytokines that target B cells. Recent progress in protein engineering may enable us to generate novel biologic therapies based on TGF-β family cytokines.

Species-Specific Chromosome Engineering Greatly Improves Fully Human Polyclonal Antibody Production Profile in Cattle.

Large-scale production of fully human IgG (hIgG) or human polyclonal antibodies (hpAbs) by transgenic animals could be useful for human therapy. However, production level of hpAbs in transgenic animals is generally very low, probably due to the fact that evolutionarily unique interspecies-incompatible genomic sequences between human and non-human host species may impede high production of fully hIgG in the non-human environment. To address this issue, we performed species-specific human artificial chromosome (HAC) engineering and tested these engineered HAC in cattle. Our previous study has demonstrated that site-specific genomic chimerization of pre-B cell receptor/B cell receptor (pre-BCR/BCR) components on HAC vectors significantly improves human IgG expression in cattle where the endogenous bovine immunoglobulin genes were knocked out. In this report, hIgG1 class switch regulatory elements were subjected to site-specific genomic chimerization on HAC vectors to further enhance hIgG expression and improve hIgG subclass distribution in cattle. These species-specific modifications in a chromosome scale resulted in much higher production levels of fully hIgG of up to 15 g/L in sera or plasma, the highest ever reported for a transgenic animal system. Transchromosomic (Tc) cattle containing engineered HAC vectors generated hpAbs with high titers against human-origin antigens following immunization. This study clearly demonstrates that species-specific sequence differences in pre-BCR/BCR components and IgG1 class switch regulatory elements between human and bovine are indeed functionally distinct across the two species, and therefore, are responsible for low production of fully hIgG in our early versions of Tc cattle. The high production levels of fully hIgG with hIgG1 subclass dominancy in a large farm animal species achieved here is an important milestone towards broad therapeutic applications of hpAbs.

Chronic upper airway inflammation is characterized by an extrafollicular B cell response and elevated antibody production in humans (HUM1P.260)

Abstract Chronic rhinosinusitis (CRS) is an upper airway disease that affects 30 million Americans and a subgroup of CRS patients also has nasal polyps (CRSwNP). In CRSwNP patients, inflamed sinus tissues can be removed as part of treatment, which provides a unique model system to investigate mechanisms of human mucosal inflammation. We have previously reported local elevations in NP of B cells, antibodies (Ab), and expression of Epstein-Barr virus-Induced protein 2 (EBI2), a marker of extrafollicular plasma cells (PC). To characterize the mechanisms mediating these responses, we assessed B cells from fresh tissue (d0) or after 4 days of culture (d4) from NP, uncinate tissue (UT) or tonsils from CRS and control subjects by flow cytometry. We measured Ab production by multiplex array and assessed formation of B cell follicles by immunohistochemistry. We assessed the Ab repertoire by DNA deep sequencing and by analysis of Ab genes from single cells. We found that NP uniquely supported the accumulation of PC (p<0.01) and Ab production (p<0.05) in vitro. Plasmablasts (PB) and PC were Ki67+, and EBI2 expression was highest on PB. We found no evidence for increased B cell follicles in NP compared to control UT (n>10/group). Ab from NP had fewer CDR3 mutations than Ab from UT (p<0.05), but more mutations than Ab from peripheral blood (p<0.05). These data suggest that there is a strong extrafollicular B cell response in NP that may drive Ab production during chronic airway inflammation.

The Usefulness of an Influenza Virus-Like Particle (VLP) Vaccine Produced in Silkworm Pupae and Virosomes and Liposomes Prepared by Chemical Means: From Virosome to VLP and the Future of Vaccines

In our quest for an ideal influenza vaccine in 1989, we successfully prepared a muramyl dipeptide (MDP)-liposome-containing influenza virus hemagglutinin (HA) and neuraminidase (NA) virosome vaccine. This MDP-virosome vaccine showed enhanced HA immunogenicity in mice and elicited superior antibody production in humans, and a high HI titer was retained for up to 2 years. Generation of a cellular immune response was also confirmed by the production of cytotoxic T lymphocytes in mice. In fact, these killer T cells reduced the titer of the H3N2, H1N1 and B virus in the lungs of infected mice. Based on the finding that this MDP-virosome vaccine could elicit both humoral and cellular immune responses (i.e., high antibody activity and killer T cells), which is a requirement for an ideal vaccine, we examined the possibility of preparing the VLP vaccine in silkworms. Here, we show that significant amounts of influenza VLPs could be successfully produced in silkworm pupae by using a chimeric (multiple hybrid) HA DNA. In fact, the HA titers in homogenates from the infected pupae reached a mean value of 0.8 million HA units (HAU), equivalent to approximately 2,000 μg of HA protein per pupa, which is more than 50-fold higher than that produced in embryonated chicken eggs. Analysis of the VLPs in the purified HA fractions showed that they were attractive structures ranging from 30 to 300 nm in diameter. The spikes on the surface of the VLPs were approximately 14 nm long, densely packed, and were very similar to those on actual influenza virus particles. Subsequent important evidence was symbolized in the following genome constellation. In the present study, we showed, for the first time, that VLPs could be synthesized in silkworms by injection of a single synthetic chimeric HA DNA. These VLPs reflected the characteristic structure of the influenza virosome and elicited immune responses that were much higher than those elicited by the MDP-liposome prepared in our previous study. Therefore, the use of chemical immunomodulators such as MDP should be considered in the future for the development of VLP vaccines.

Use of Yeast Lysate in Women with Recurrent Vulvovaginal Candidiasis

Vulvovaginal candidiasis (VVC) affects a significant number of women, especially in working age. In an estimated 75% of women an episode of acute vulvovaginal candidiasis occurs during lifetime and another 5–10% of women develop recurrent vulvovaginal candidiasis (RVVC). This is mainly characterized by intense burning, itching, pain, abnormal discharge, dyspareunia. Immune response to candidiasis is both cellular (CMI) (natural protection mechanisms) and humoral (antibody production). Understanding the principles of immunity in candidiasis is also important for development of candida vaccines.CANDIVAC contains lyophilized Candida lysate (C. albicans, C. krusei, C. glabrata) together with immunostimulatory bacterial strain of Propionibacterium acnes. The product is taken orally in capsules for 10 days followed by a 20-day pause. It is administered for 3 to 6 months. The product has been tested in a total of 75 women at the age of 18–45 years. In these women at least 4 episodes of vulvovaginal candidiasis have been microscopically or laboratory diagnosed during the last 12 months. Following CANDIVAC administration, statistically significant changes occurred in the evaluation of subjective and some objective criteria. The most important marker of product efficiency is a significant reduction in recurrence compared to the recent state. This criterion has a fundamental importance in patient satisfaction. Before medication the patients suffered from at least 4 attacks, while after medication an attack occurred in only 31% of women and more than 2 attacks in only 3% of treated women.Compromised balance of immune system plays a major role in recurrent vulvovaginal candidiasis. Specific oral product CANDIVAC, prepared from the most common strains of yeast infections, supports immune mechanisms, ensuring resistance of the human organism against yeasts. Its administration significantly prolongs remission, leads to a reduction in application of antimycotics and also changes properties of cellular and humoral immunity in medicated patients.

Human antibody production in transgenic animals.

Fully human antibodies from transgenic animals account for an increasing number of new therapeutics. After immunization, diverse human monoclonal antibodies of high affinity can be obtained from transgenic rodents, while large animals, such as transchromosomic cattle, have produced respectable amounts of specific human immunoglobulin (Ig) in serum. Several strategies to derive animals expressing human antibody repertoires have been successful. In rodents, gene loci on bacterial artificial chromosomes or yeast artificial chromosomes were integrated by oocyte microinjection or transfection of embryonic stem (ES) cells, while ruminants were derived from manipulated fibroblasts with integrated human chromosome fragments or human artificial chromosomes. In all strains, the endogenous Ig loci have been silenced by gene targeting, either in ES or fibroblast cells, or by zinc finger technology via DNA microinjection; this was essential for optimal production. However, comparisons showed that fully human antibodies were not as efficiently produced as wild-type Ig. This suboptimal performance, with respect to immune response and antibody yield, was attributed to imperfect interaction of the human constant region with endogenous signaling components such as the Igα/β in mouse, rat or cattle. Significant improvements were obtained when the human V-region genes were linked to the endogenous CH-region, either on large constructs or, separately, by site-specific integration, which could also silence the endogenous Ig locus by gene replacement or inversion. In animals with knocked-out endogenous Ig loci and integrated large IgH loci, containing many human Vs, all D and all J segments linked to endogenous C genes, highly diverse human antibody production similar to normal animals was obtained.

Principles and application of antibody libraries for infectious diseases.

Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombinant antibody technology allows the generation of fully human antibodies. Phage display is the gold standard for the production of human antibodies in vitro. To generate monoclonal antibodies by phage display, the generation of antibody libraries is crucial. Antibody libraries are classified according to the source where the antibody gene sequences were obtained. The most useful library for infectious diseases is the immunized library. Immunized libraries would allow better and selective enrichment of antibodies against disease antigens. The antibodies generated from these libraries can be translated for both diagnostic and therapeutic applications. This review focuses on the generation of immunized antibody libraries and the potential applications of the antibodies derived from these libraries.

Correction: Triple Immunoglobulin Gene Knockout Transchromosomic Cattle: Bovine Lambda Cluster Deletion and Its Effect on Fully Human Polyclonal Antibody Production

Correction: Triple Immunoglobulin Gene Knockout Transchromosomic Cattle: Bovine Lambda Cluster Deletion and Its Effect on Fully Human Polyclonal Antibody Production

Phosphoproteomic analyses reveal that galectin-1 augments the dynamics of B-cell receptor signaling

B-cell activation is important for mounting humoral immune responses and antibody production. Galectin-1 has multiple regulatory functions in immune cells. However, the effects of galectin-1 modulation and the mechanisms underlying the coordination of B-cell activation are unclear. To address this issue, we applied label-free quantitative phosphoproteomic analysis to investigate the dynamics of galectin-1-induced signaling in comparison with that following anti-IgM treatment. A total of 3247 phosphorylation sites on 1245 proteins were quantified, and 70-80% of the 856 responsive phosphoproteins were commonly activated during various biological functions. The similarity between galectin-1- and anti-IgM-elicited B-cell receptor (BCR) signaling pathways was also revealed. Additionally, the mapping of the 149 BCR-responsive phosphorylation sites provided complementary knowledge of BCR signaling. Compared to anti-IgM induction, the phosphoproteomic profiling of BCR signaling, along with validation by western blot analysis and pharmacological inhibitors, revealed that the activation of Syk, Btk, and PI3K may be dominant in galectin-1-mediated activation. We further demonstrated that the proliferation of antigen-primed B cells was diminished in the absence of galectin-1 in an animal model. Together, these findings provided evidence for a new role and insight into the mechanism of how galectin-1 augments the strength of the immunological synapse by modulating BCR signaling. The current study revealed the first systematic phosphorylation-mediated signaling network and its dynamics in B cell activation. The comparative phosphoproteomic analysis on the dynamics of galectin-1 induced activation profiles not only showed that exogenously added galectin-1 augmented B-cell activation but also revealed its relatively enhanced activation in PI3K pathway. Together with proliferation assay, we further delineated that galectin-1 is important for B-cell proliferation in response to antigen challenge. Our phosphoproteomic study reveals a new role for galectin-1 in augmenting the strength of immunological synapse by modulating BCR signaling.

Detection of Influenza A/H1N1-Specific Human IgG-Secreting B Cells in Older Adults by ELISPOT Assay

B cells play an important role in humoral immunity and antibody production. Use of a B cell ELISPOT assay to quantify antigen-specific B cells can assist other assays to achieve a more complete profile of the humoral immune response after vaccination. We utilized a B cell ELISPOT assay to measure the number of influenza A/H1N1-specific B cells at key timepoints after seasonal influenza vaccination in 106 older adults (50-74 years of age). Blood was drawn from these subjects on Day 0, Day 3, Day 28, and Day 75 after vaccination to represent baseline, early, peak, and late response, respectively, of influenza A/H1N1-specific B cells. A significant increase in A/H1N1-specific B cells (median 36 spot-forming units/SFUs per 200,000 cells, p<0.0001) was seen on Day 28 compared to baseline and Day 3, and this number decreased (23 SFUs, p<0.0001) by Day 75, but not to baseline level. These data suggest that the B cell ELISPOT can be used to profile and monitor the humoral immune responses in older subjects after influenza vaccination, and serve as an immune signature marker.

Production of human antibodies by in vitro immunization using a fusion protein containing the transcriptional transactivator of HIV-1

Antigen-specific activation of human B cells represents a key step for the production of monoclonal antibodies. Several approaches have been developed over the last thirty years in order to improve the process of lymphocyte activation in vitro. In the present study, we investigated whether the transcriptional transactivator (Tat) of human immunodeficiency virus, which possesses numerous biological activities, is able to trigger antibody secretion when incubated with human peripheral blood mononuclear cells. No such effect was observed when using Tat as a free protein. However, we found a significant IgM antibody production when Tat was previously fused to a double domain, called ZZ, derived from protein A of Staphylococcus aureus. The effect was also observed when the fusion protein, called ZZTat101, was incubated with purified B cells, indicating that the phenomenon does not require T-cell help. Antibody secretion was observed in the absence of cytokines that are usually used during in vitro immunization experiments, indicating that ZZTat101 provides the signals required for the initiation of the immune response. Antibody secretion was observed using a ZZTat mutant, containing only the Tat residues 22 to 57, called ZZTat22–57, indicating that this region is sufficient to initiate the immune response. In contrast, the effect was not found with a ZZTat22–57 mutant devoid of the seven Tat cysteines located between residues 22 and 37, demonstrating that these residues play a crucial role in the phenomenon. Our results pave the way to the development of a new in vitro immunization method based on antigens associated with ZZTat.