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Radioprotective 105 (RP105) (also termed CD180) is an orphan and unconventional Toll-like receptor (TLR) that lacks an intracellular signaling domain. The agonistic anti-RP105 monoclonal antibody (mAb) can cross-link RP105 on B cells, resulting in the proliferation and activation of B cells. Anti-RP105 mAb also has a potent adjuvant effect, providing higher levels of antigen-specific antibodies compared to alum. However, adjuvanticity is required for the covalent link between anti-RP105 mAb and the antigen. This is a possible obstacle to immunization due to the link between anti-RP105 mAb and some antigens, especially multi-transmembrane proteins. We have previously succeeded in inducing rapid and potent recombinant mAbs in mice using antibody gene-based delivery. To simplify the covalent link between anti-RP105 mAb and antigens, we generated genetic constructs of recombinant anti-RP105 mAb (αRP105) bound to the transmembrane domain of the IgG-B cell receptor (TM) (αRP105-TM), which could enable the anti-RP105 mAb to link the antigen via the cell membrane. We confirmed the expression of αRP105-TM and the antigen hemagglutinin, which is a membrane protein of the influenza virus, on the same cell. We also found that αRP105-TM could activate splenic B cells, including both mature and immature cells, depending on the cell surface RP105 in vitro . To evaluate the adjuvanticity of αRP105-TM, we conducted DNA immunization in mice with the plasmids encoding αRP105-TM and hemagglutinin, followed by challenge with an infection of a lethal dose of an influenza virus. We then obtained partially but significantly hemagglutinin-specific antibodies and observed protective effects against a lethal dose of influenza virus infection. The current αRP105-TM might provide adjuvanticity for a vaccine via a simple preparation of the expression plasmids encoding αRP105-TM and of that encoding the target antigen., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Yamazaki, Biswas, Kosugi, Nagashima, Inui, Tomono, Takagi, Ichimonji, Nagaoka, Ainai, Hasegawa, Chiba and Akashi-Takamura.)

Influenza is an infectious respiratory illness caused by influenza viruses. Despite yearly updates, the efficacy of influenza vaccines is significantly curtailed by the virus antigenic drift and antigenic shift. These constant changes to the influenza virus make-up also challenge the development of a universal flu vaccine, which requires conserved antigenic regions shared by influenza viruses of different subtypes. We propose that it is possible to bypass these challenges by the development of an influenza vaccine based on conserved proteins delivered in an adjuvanted nanoparticle system. In this study, we generated influenza nanoparticle constructs using trimethyl chitosan nanoparticles (TMC nPs) as the carrier of recombinant influenza hemagglutinin subunit 2 (HA2) and nucleoprotein (NP). The purified HA2 and NP recombinant proteins were encapsulated into TMC nPs to form HA2-TMC nPs and NP-TMC nPs, respectively. Primary human intranasal epithelium cells (HNEpCs) were used as an in vitro model to measure immunity responses. HA2-TMC nPs, NP-TMC nPs, and HA2-NP-TMC nPs (influenza nanoparticle constructs) showed no toxicity in HNEpCs. The loading efficiency of HA2 and NP into the TMC nPs was 97.9% and 98.5%, respectively. HA2-TMC nPs and NP-TMC nPs more efficiently delivered HA2 and NP proteins to HNEpCs than soluble HA2 and NP proteins alone. The induction of various cytokines and chemokines was more evident in influenza nanoparticle construct-treated HNEpCs than in soluble protein-treated HNEpCs. In addition, soluble factors secreted by influenza nanoparticle construct-treated HNEpCs significantly induced MoDCs maturation markers (CD80, CD83, CD86 and HLA-DR), as compared to soluble factors secreted by protein-treated HNEpCs. HNEpCs treated with the influenza nanoparticle constructs significantly reduced influenza virus replication in an in vitro challenge assay. The results indicate that TMC nPs can be used as influenza vaccine adjuvants and carriers capable of delivering HA2 and NP proteins to HNEpCs., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Influenza is a severe viral disease, a frequent complication of which is a secondary bacterial pneumonia. Influenza vaccines prevent secondary bacterial complications. Virus-like particles are one of the promising areas for the development of new vaccines. The aim of this work is to study the correlation of the pathomorphological characteristics of the lungs with clinical, virological, and microbiological markers of the disease at vaccination with virus-like particles (VLPs), containing hemagglutinin (HA) of influenza virus (HA-Gag-VLPs) in a murine model of secondary bacterial pneumonia induced by S. pneumoniae after influenza infection., Material and Methods: BALB/c mice were vaccinated with VLPs containing influenza HA. After 21 days, mice were infected with two strains of influenza viruses, homologous and non-homologous, and 5 days after viral infection, were infected with S. pneumoniae. The vaccination effect was evaluated by morphological, virological (titer of the virus in the lungs) and microbiological (titer of bacteria in the lungs) data, and was confirmed by clinical data (survival, change in body weight)., Results: Immunization with HA-Gag-VLPs, followed by infection with a homologous influenza virus and S. pneumoniae, reduced the area of foci of inflammation, inhibited the replication of the virus and bacteria in the lungs, and also protected animals from death and reduced their weight loss. Immunization with HA-Gag-VLPs upon infection with a heterologous strain and S. pneumoniae did not affect these criteria., Conclusion: The immunization with HA-Gag-VLPs prevented the viral replication, providing a reduction of S. pneumoniae titer and the degree of lung damage, protecting animals from the disease in a murine model of secondary bacterial pneumonia, induced by S. pneumoniae, after influenza infection with homologous strain of the virus.

Protein-based, self-assembling nanoparticles elicit superior immunity compared with soluble protein vaccines, but the immune mechanisms underpinning this effect remain poorly defined. Here, we investigated the immunogenicity of a prototypic ferritin-based nanoparticle displaying influenza hemagglutinin (HA) in mice and macaques. Vaccination of mice with HA-ferritin nanoparticles elicited higher serum antibody titers and greater protection against experimental influenza challenge compared with soluble HA protein. Germinal centers in the draining lymph nodes were expanded and persistent following HA-ferritin vaccination, with greater deposition of antigen that colocalized with follicular dendritic cells. Our findings suggest that a highly ordered and repetitive antigen array may directly drive germinal centers through a B cell-intrinsic mechanism that does not rely on ferritin-specific T follicular helper cells. In contrast to mice, enhanced immunogenicity of HA-ferritin was not observed in pigtail macaques, where antibody titers and lymph node immunity were comparable to soluble vaccination. An improved understanding of factors that drive nanoparticle vaccine immunogenicity in small and large animal models will facilitate the clinical development of nanoparticle vaccines for broad and durable protection against diverse pathogens.

This study aimed to investigate whether the human antigen presenting cells (APCs) can process and present Salmonella expressing H7N9 hemagglutinin (Sal-HA), neuraminidase (Sal-NA) or M2 ectodomain (Sal-M2e) to T cells and subsequently activate CD4 + T cell responses in vitro. In this study, APCs generated from human peripheral blood mononuclear cells (PBMCs) were first treated with mitomycin-C, followed by stimulation with Sal-HA, Sal-M2e, Sal-NA or Salmonella alone for 24 h. Subsequently, stimulated APCs were coincubated with untreated PBMCs (1:10) of the same individual for 24 or 72 h and then analysed for cytokine induction and T cell proliferations by qRT-PCR assay and flow cytometry, respectively. Our results demonstrated that APCs stimulated with Sal-HA, Sal-M2e or Sal-NA induced significantly (p < .05) higher CD3 + CD4 + T cell proliferations compared to the APCs treated with Salmonella alone. Our data further revealved that APCs treated with Sal-HA induced significantly (p < .05) higher CD3 + CD4 + T cell responses compared to the APCs treated with either Sal-M2e or Sal-NA, which both induced almost comparable levels. The T cell proliferation responses were further measured by lymphocyte proliferation assay and the results showed that Sal-HA and Sal-M2e stimulated APCs induced significantly (p < .05) higher proliferations in T cells compared to the APCs stimulated with either Sal-NA or Salmonella alone. With respect to cytokine inductions, APCs treated with either Sal-HA or Sal-M2e induced significantly (p < .05) higher mRNA transcription levels of proinflammatory (IL-1β, IL-6, IL-12 and IL-23), Th1 (IFN-γ), Th17 (IL-17 and IL-21) and Th2 (IL-10 and TGF-β) cytokines in T cells compared to Sal-NA or Salmonella alone treated APCs. In conclusion, we show that Salmonella system can efficiently deliver vaccine antigens to APCs and is, thus, capable to elicit heterologous antigen-specific adaptive immunity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Hemagglutinin (HA) displayed on a ferritin nano-cage has been shown to be effective in generating a potent immune response against a broad range of influenza infections. Here, we showed that conjugation of flagellin together with HA to the exterior surface of the ferritin cage greatly enhanced not only the humoral immune response in mice but also antigen-specific T cell responses that include Th1 cytokine secretion. The effect of flagellin remained essentially unchanged when the molar ratio of flagellin to HA was reduced from 1:1 to 1:3. Injection of the ferritin-HA-flagellin cage provided protection against lethal virus challenge in mice. We used a small immunoglobulin fragment V L 12.3 as a convenient method for attaching HA and flagellin to the ferritin cage. This attachment method can be used for rapid screening of a variety of protein cages and nano-assemblies to identify the most suitable carrier and adjuvant proteins for the target antigen., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Swine influenza viruses (SIVs), the causal agents of swine influenza, are not only important to control due to the economic losses in the swine industry, but also can be pandemic pathogens. Vaccination is one of the most relevant strategies to control and prevent influenza infection. Current human vaccines against influenza induce strain-specific immunity and annual update is required due to the virus antigenic shift phenomena. Previously, our group has reported the use of conserved hemagglutinin peptides (HA-peptides) derived from H1-influenza virus as a potential multivalent vaccine candidate. Immunization of swine with these HA-peptides elicited antibodies that recognized and neutralized heterologous influenza viruses in vitro and demonstrated strong hemagglutination-inhibiting activity. In the present work, we cloned one HA-peptide (named NG34) into a plasmid fused with cytotoxic T lymphocyte-associated antigen (CTLA4) which is a molecule that modifies T cell activation and with an adjuvant activity interfering with the adaptive immune response. The resulting plasmid, named pCMV-CTLA4-Ig-NG34, was administered twice to animals employing a needle-free delivery approach. Two studies were carried out to test the efficacy of pCMV-CTLA4-Ig-NG34 as a potential swine influenza vaccine, one in seronegative and another in seropositive pigs against SIV. The second one was aimed to evaluate whether pCMV-CTLA4-Ig-NG34 vaccination would overcome maternally derived antibodies (MDA). After immunization, all animals were intranasally challenged with an H3N2 influenza strain. A complete elimination or significant reduction in the viral shedding was observed within the first week after the challenge in the vaccinated animals from both studies. In addition, no challenged heterologous virus load was detected in the airways of vaccinated pigs. Overall, it is suggested that the pCMV-CTLA4-Ig-NG34 vaccine formulation could potentially be used as a multivalent vaccine against influenza viruses., Competing Interests: The authors have declared that no competing interests exist.

Due to the high risk of an outbreak of pandemic influenza, the development of a broadly protective universal influenza vaccine is highly warranted. The design of such a vaccine has attracted attention and much focus has been given to nanoparticle-based influenza vaccines which can be administered intranasally. This is particularly interesting since, contrary to injectable vaccines, mucosal vaccines elicit local IgA and lung resident T cell immunity, which have been found to correlate with stronger protection in experimental models of influenza virus infections. Also, studies in human volunteers have indicated that pre-existing CD4 + T cells correlate well to increased resistance against infection. We have previously developed a fusion protein with 3 copies of the ectodomain of matrix protein 2 (M2e), which is one of the most explored conserved influenza A virus antigens for a broadly protective vaccine known today. To improve the protective ability of the self-adjuvanting fusion protein, CTA1-3M2e-DD, we incorporated it into porous maltodextrin nanoparticles (NPLs). This proof-of-principle study demonstrates that the combined vaccine vector given intranasally enhanced immune protection against a live challenge infection and reduced the risk of virus transmission between immunized and unimmunized individuals. Most importantly, immune responses to NPLs that also contained recombinant hemagglutinin (HA) were strongly enhanced in a CTA1-enzyme dependent manner and we achieved broadly protective immunity against a lethal infection with heterosubtypic influenza virus. Immune protection was mediated by enhanced levels of lung resident CD4 + T cells as well as anti-HA and -M2e serum IgG and local IgA antibodies.

Background: Influenza infection could be more effectively controlled if a multi-purpose vaccine with the ability to induce responses against most, or all, influenza A subtypes could be generated. Conserved viral proteins are a promising basis for the creation of a broadly protective vaccine. In the present study, the immunogenicity and protective properties of three recombinant proteins (vaccine candidates), comprising conserved viral proteins fused with bacterial flagellin, were compared., Methods: Balb/c mice were immunized intranasally with recombinant proteins comprising either one viral protein (the ectodomain of the M2 protein, 'M2e') or two viral proteins (M2e and the hemagglutinin second subunit 'HA2' epitope) genetically fused with flagellin. Further, two different consensus variants of HA2 were used. Therefore, three experimental positives were used in addition to the negative control (Flg-his). The mucosal, humoral, and T-cell immune responses to these constructs were evaluated., Result: We have demonstrated that insertion of the HA2 consensus polypeptide (aa 76-130), derived from either the first (HA2-1) or second (HA2-2) virus phylogenetic group, into the recombinant Flg4M2e protein significantly enhanced its immunogenicity and protective properties. Intranasal administration of the vaccine candidates (Flg-HA2-2-4M2e or Flg-HA2-1-4M2e) induced considerable mucosal and systemic responses directed at both the M2e-protein and, in general, the influenza A virus. However, the immune response elicited by the Flg-HA2-1-4M2e protein was weaker than the one generated by Flg-HA2-2-4M2e. These recombinant proteins containing both viral peptides provide complete protection from lethal challenge with various influenza viruses: A/H3N2; A/H2N2; and A/H5N1., Conclusion: This study demonstrates that the intranasal administration of Flg-HA2-2-4M2e recombinant protein induces a strong immune response which provides broad protection against various influenza viruses. This construct is therefore a strong candidate for development as a universal vaccine., Competing Interests: The authors have declared that no competing interests exist.

Seasonal influenza is a contagious respiratory illness that annually affects millions of people worldwide. To identify currently circulating influenza virus subtypes, the Centers for Disease Control and Prevention's International Reagent Resource distributes the World Health Organization (WHO) influenza reagent kits, which are used globally by testing laboratories for influenza surveillance. The data generated by the kits aid in strain selection for the influenza vaccine each season. The use of animals to produce high quality and quantities of antibodies is critical to the production of these kits. In this study, we assessed the effects and efficacy of repeated sampling from automated plasmapheresis in goats. Analysis of blood samples demonstrated that repeated automated plasmapheresis procedures did not adversely affect the immediate or long-term health of goats. Further, our results indicate that repeated plasmapheresis in goats was capable of generating 2 liters of antibody-rich plasma per goat per week. This volume is sufficient to produce enough WHO influenza kits to conduct over 1 million tests. Thus, we have shown that the rapid production of plasma in goats can positively impact the public health preparedness and response to influenza., Competing Interests: The authors have declared that no competing interests exist.

Health policy precludes neonatal vaccination against influenza. Hence, morbidity and mortality are high under 6 months of age. Lactoferrin may activate diminished numbers of dysfunctional dendritic cells and reverse neonatal vaccine failures. Aluminum hydroxide/ALUM recruits neutrophils that secrete lactoferrin at deposition sites of antigen. We theorized lactoferrin + influenza antigen initiates an equivalent antibody response compared to ALUM. Three-day-old mice received subcutaneously 30 μg of H1N1 hemagglutinin + 200 μg of bovine lactoferrin versus hemagglutinin + ALUM. Controls received hemagglutinin, lactoferrin, or ALUM. After 21 days, sera measured anti-H1N1 (ELISA) and neutralizing antibody (plaque assays). ELISA detected equal antibody production with lactoferrin + hemagglutinin compared to hemagglutinin + ALUM; both sera also neutralized H1N1 virus at a 1:20 dilution (p < 0.01). Controls had no anti-H1N1 antibody. Neonates given lactoferrin had no anaphylaxis when challenged four weeks later. Lactoferrin is a safe and effective adjuvant for inducing antibody against influenza in neonates., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Dendritic cells are major APCs that can efficiently prime immune responses. However, the roles of skin-resident Langerhans cells (LCs) in eliciting immune responses have not been fully understood. In this study, we demonstrate for the first time, to our knowledge, that LCs in cynomolgus macaque skin are capable of inducing antiviral-specific immune responses in vivo. Targeting HIV-Gag or influenza hemagglutinin Ags to skin LCs using recombinant fusion proteins of anti-Langerin Ab and Ags resulted in the induction of the viral Ag-specific responses. We further demonstrated that such Ag-specific immune responses elicited by skin LCs were greatly enhanced by TLR ligands, polyriboinosinic polyribocytidylic acid, and R848. These enhancements were not due to the direct actions of TLR ligands on LCs, but mainly dependent on TNF-α secreted from macrophages and neutrophils recruited to local tissues. Skin LC activation and migration out of the epidermis are associated with macrophage and neutrophil infiltration into the tissues. More importantly, blocking TNF-α abrogated the activation and migration of skin LCs. This study highlights that the cross-talk between innate immune cells in local tissues is an important component for the establishment of adaptive immunity. Understanding the importance of local immune networks will help us to design new and effective vaccines against microbial pathogens., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The recombinant hemagglutinin (rHA)-based influenza vaccine Flublok® has recently been approved in the United States as an alternative to the traditional egg-derived flu vaccines. Flublok is a purified vaccine with a hemagglutinin content that is threefold higher than standard inactivated influenza vaccines. When rHA derived from an H3N2 influenza virus was expressed, purified, and stored for 1 month, a rapid loss of in vitro potency (∼50%) was observed as measured by the single radial immunodiffusion (SRID) assay. A comprehensive characterization of the rHA protein antigen was pursued to identify the potential causes and mechanisms of this potency loss. In addition, the biophysical and chemical stability of the rHA in different formulations and storage conditions was evaluated over time. Results demonstrate that the potency loss over time did not correlate with trends in changes to the higher order structure or hydrodynamic size of the rHA. The most likely mechanism for the early loss of potency was disulfide-mediated cross-linking of rHA, as the formation of non-native disulfide-linked multimers over time correlated well with the observed potency loss. Furthermore, a loss of free thiol content, particularly in specific cysteine residues in the antigen's C-terminus, was correlated with potency loss measured by SRID., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

The 2009 H1N1 pandemic and recent human cases of H5N1, H7N9, and H6N1 in Asia highlight the need for a universal influenza vaccine that can provide cross-strain or even cross-subtype protection. Here, we show that recombinant monoglycosylated hemagglutinin (HAmg) with an intact protein structure from either seasonal or pandemic H1N1 can be used as a vaccine for cross-strain protection against various H1N1 viruses in circulation from 1933 to 2009 in mice and ferrets. In the HAmg vaccine, highly conserved sequences that were originally covered by glycans in the fully glycosylated HA (HAfg) are exposed and thus, are better engulfed by dendritic cells (DCs), stimulated better DC maturation, and induced more CD8+ memory T cells and IgG-secreting plasma cells. Single B-cell RT-PCR followed by sequence analysis revealed that the HAmg vaccine activated more diverse B-cell repertoires than the HAfg vaccine and produced antibodies with cross-strain binding ability. In summary, the HAmg vaccine elicits cross-strain immune responses that may mitigate the current need for yearly reformulation of strain-specific inactivated vaccines. This strategy may also map a new direction for universal vaccine design.

The modern influenza virus subtypes H3N2, H5N1, and H1N1 reduced the metabolism of the endothelial cells within the range from 20% to 60% (compared with control). The degree of the activity of the dehydrogenase reduction depended on the dose of virus and time of virus reproduction. HA and NA also actively reduced the metabolism of the cells ranging from 5% to 60%, depending on the concentration of the proteins and time of their impact on cells. Neuraminidase was more active than hemagglutinin in the MTT test (at concentration 50 microg protein/ml).

Major histocompatibility complex (MHC) class II-associated antigen presentation involves an array of interacting molecules. CD74, the cell surface isoform of the MHC class II-associated invariant chain, is one such molecule; its role remains poorly defined. To address this, we have employed a high-resolution single-particle imaging method for quantifying the colocalization of CD74 with human leukocyte antigen (HLA)-DR molecules on human fibroblast cells known for their capacity to function as antigen-presenting cells. We have also examined whether the colocalization induces internalization of HLA-DR using HA(307-319), a "universal" peptide that binds specifically to the peptide-binding groove of all HLA-DR molecules, irrespective of their alleles. We have determined that 25 ± 1.3% of CD74 and 17 ± 0.3% of HLA-DR are colocalized, and the association of CD74 with HLA-DR and the internalization of HLA-DR are both inhibited by HA(307-319). A similar inhibition of HLA-DR internalization was observed in freshly isolated monocyte-derived dendritic cells. A key role of CD74 is to translocate HLA-DR molecules to early endosomes for reloading with peptides prior to recycling to the cell surface. We conclude that CD74 regulates the balance of peptide-occupied and peptide-free forms of MHC class II at the cell surface.

The threat of a new influenza pandemic has existed since 1997, when the highly pathogenic H5N1 strain of avian influenza A virus infected humans in Hong Kong and spread across Asia, where it continued to infect poultry and people. The human mortality rate of H5N1 infection is about 60%, whereas that of seasonal H1N1 infection is less than 0.1%. The high mortality rate associated with H5N1 infection is predominantly a result of respiratory failure caused by acute lung injury; however, how viral infection contributes to this disease pathology is unclear. Here, we used electron microscopy to show the accumulation of autophagosomes in H5N1-infected lungs from a human cadaver and mice, as well as in infected A549 human epithelial lung cells. We also showed that H5N1, but not seasonal H1N1, induced autophagic cell death in alveolar epithelial cells through a pathway involving the kinase Akt, the tumor suppressor protein TSC2, and the mammalian target of rapamycin. Additionally, we suggest that the hemagglutinin protein of H5N1 may be responsible for stimulating autophagy. When applied prophylactically, reagents that blocked virus-induced autophagic signaling substantially increased the survival rate of mice and substantially ameliorated the acute lung injury and mortality caused by H5N1 infection. We conclude that the autophagic cell death of alveolar epithelial cells likely plays a crucial role in the high mortality rate of H5N1 infection, and we suggest that autophagy-blocking agents might be useful as prophylactics and therapeutics against infection of humans by the H5N1 virus.

There has been an increase in interest in the use of altered peptides as antigen-specific therapeutic agents in autoimmune diseases. Here we investigated the inhibitory effect and possible mechanism of an altered influenza virus haemagglutinin (HA)-derived peptide in collagen-induced arthritis (CIA). CIA was induced in DBA/1 mice by immunisation with type II collagen (CII). Altered HA308-317, wild-type HA308-317 or irrelevant peptide was administered intranasally beginning from arthritis onset. Clinical and histological scores were assessed, and cytokine levels in the serum or supernatants from splenocytes were determined. The percentages of Th1 and Th2 cells in response to different peptides were analysed by FACS both in vivo and in vitro. Our results showed that intranasal administration of altered HA308-317 peptide significantly ameliorated CIA. The therapeutic effect of altered HA308-317 peptide was associated with a substantial decrease in production of interferon (IFN)-γ, interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, anti-CII IgG, IgG1 and IgG2a antibodies, and an markedly increase in production of IL-10 and IL-4 in serum or supernatants from splenocytes treated with altered HA308-317 peptide. The percentage of Th2 (CD4(+)IL-4(+)) cells was upregulated significantly by altered HA308-317 peptide with a decreased percentage of Th1 (T helper 1; CD4(+)INF-γ(+)) cells both in vivo and in vitro. These findings suggest that altered HA308-317 peptide might be a promising candidate for rheumatoid arthritis (RA) treatment.

Despite its use widely as a therapeutic agent, and proposed use as vaccine adjuvant, the effect of IFNalpha on T cell function is poorly understood. As a pleiotropic innate cytokine produced rapidly in response to pathogens, it is well placed to impinge on specific immune responses. The aim of this study was to examine the impact of IFNalpha on the function of human memory CD4(+) T cells using the recall Ags purified protein derivative, tetanus toxoid, and hemagglutinin. IFNalpha administered either in vivo or added exogenously in vitro tended to enhance proliferative responses of purified protein derivative-specific T cells in marked contrast to the other cognate populations whose responses were often diminished. Purifying the memory CD4(+)CD45RO(+) T cells confirmed IFNalpha acted directly on these cells and not via an intermediate. The T cells could be divided into two broad categories depending on how IFNalpha effected their responses to cognate Ag: 1) enhanced proliferation and a striking increase in IFNgamma-production compared with smaller increases in IL-10 (increased ratio of IFNgamma:IL-10), and 2) neutral or diminished proliferation coupled with a smaller increase in IFNgamma relative to the increase in IL-10 (reduced IFNgamma:IL-10 ratio). IFNalpha has a role in modifying memory T cell responses when they are exposed to cognate Ag and may be important in vaccination strategies designed to augment particular Th memory responses.

Little is known about the mechanisms of cell-cell fusion in development and diseases and, especially, about fusion stages downstream of an opening of nascent fusion pore(s). Earlier works on different cell-cell fusion reactions have indicated that cytoskeleton plays important role in syncytium formation. However, due to complexity of these reactions and multifaceted contributions of cytoskeleton in cell physiology, it has remained unclear whether cytoskeleton directly drives fusion pore expansion or affects preceding fusion stages. Here we explore cellular reorganization associated with fusion pore expansion in syncytium formation using relatively simple experimental system. Fusion between murine embryonic fibroblasts NIH3T3-based cells is initiated on demand by well-characterized fusogen influenza virus hemagglutinin. We uncouple early fusion stages dependent on protein fusogens from subsequent fusion pore expansion stage and establish that the transition from local fusion to syncytium requires metabolic activity of living cells. Effective syncytium formation for cells with disorganized actin and microtubule cytoskeleton argues against hypothesis that cytoskeleton drives fusion expansion.

In the event of another influenza virus pandemic, strategies for effective mass vaccination will urgently be needed. We used a novel transdermal patch delivery technology, known as the PassPort system, to vaccinate mice with recombinant H5 hemagglutinin with or without immunomodulators. This needle-free form of vaccine delivery induced robust serum antibody responses that were augmented by different immunomodulators that stimulated the innate immune system and protected mice against lethal challenge with a highly pathogenic avian H5N1 influenza virus.

To explore early intermediates in membrane fusion mediated by influenza virus hemagglutinin (HA) and their dependence on the composition of the target membrane, we studied lipid mixing between HA-expressing cells and liposomes containing phosphatidylcholine (PC) with different hydrocarbon chains. For all tested compositions, our results indicate the existence of at least two types of intermediates, which differ in their lifetimes. The composition of the target membrane affects the stability of fusion intermediates at a stage before lipid mixing. For less fusogenic distearoyl PC-containing liposomes at 4 degrees C, some of the intermediates inactivate, and no intermediates advance to lipid mixing. Fusion intermediates that formed for the more fusogenic dioleoyl PC-containing liposomes did not inactivate and even yielded partial lipid mixing at 4 degrees C. Thus, a more fusogenic target membrane effectively blocks nonproductive release of the conformational energy of HA. Even for the same liposome composition, HA forms two types of fusion intermediates, dissimilar in their stability and propensity to fuse. This diversity of fusion intermediates emphasizes the importance of local membrane composition and local protein concentration in fusion of heterogeneous biological membranes.

T cells undergoing lymphopenia-driven proliferation acquire effector and memory properties that can be pathogenic. Indeed, generalized lymphopenia is associated with a variety of autoimmune diseases such as type 1 diabetes. The current study was carried out to determine how CD8(+) T cells undergoing acute lymphopenic expansion respond to antigen under tolerizing conditions in vivo. Adoptive transfer of diabetes by TCR-transgenic CD8(+) T cells was enhanced following treatment of NOD. scid recipients with a high dose of soluble peptide. Furthermore, whereas TCR-transgenic CD8(+) T cells underwent clonal deletion and failed to differentiate into CTL in peptide-treated lymphoreplete recipient mice, TCR-transgenic CD8(+) T cells in a lymphopenic environment were resistant to clonal deletion, and CTL differentiation was enhanced by a high dose of soluble peptide. Moreover, peptide treatment had distinct effects on expression of the anti-apoptotic protein Bcl-X(L) in TCR-transgenic CD8(+) T cells under lymphopenic versus lymphoreplete conditions. These results demonstrate that CD8(+) T cells undergoing lymphopenia-driven expansion in NOD. scid recipients are resistant to antigen-induced tolerance, and readily differentiate into CTL upon stimulation with a high dose of soluble peptide.

Objective: To investigate whether influenza virus haemagglutinin (HA)-derived altered peptide ligands (APLs) could abrogate T-cell responses to wild type HA308-317 or type II collagen (CII) 263-272 peptides and explore the potential inhibitory effects of the altered HA308-317 peptides on T-cell activation in rheumatoid arthritis (RA)., Methods: Altered HA308-317 peptides containing substitutions of T-cell receptor (TCR)-contact residues were synthesized. Peripheral blood mononuclear cells (PBMC) were obtained from 27 HLA-DR4/1-positive RA patients. Impact of the altered HA308-317 peptides on T-cell responses and the inhibitory effects on T-cell activation were determined by using PBMC from RA., Results: The results showed that the altered HA308-317 peptides could bind to HLA-DR4/1 on cell surface and had no effects on T-cell proliferation and CD25 expression. Moreover, all the altered HA308-317 peptides inhibited T-cell proliferative responses to wild type HA308-317 or CII263-272. In addition, Th1 type cytokine profile was found when PBMC were cultured with wild type HA308-317 or CII263-272, but not the altered HA 308-317 peptides., Conclusions: It is suggested that altered HA308-317 peptides bind to the RA-associated HLA-DR4/1 with no stimulating effects on T cells and might be potentially important in inhibition of T-cell activation in RA.

Collagen (CII) 263-272 peptide, an autoantigen in rheumatoid arthritis, is a specific human leukocyte antigen (HLA)-DR1/4-binding peptide recognized by T-cell receptors (TCR). The affinity of influenza virus haemagglutinin (HA) 306-318 peptide for the antigen-binding groove of HLA-DR1/4 molecules is higher than that of CII263-272. The HLA-DR1/4-binding residues of HA306-318 are located in the region 308-317. Altered HA308-317 peptides with substitutions of TCR-contact residues may inhibit HLA-DR1/4-specific T-cell activation by blocking the antigen-binding site of HLA-DR1/4 molecules. To evaluate the role of altered HA308-317 peptides in HLA-DR1-restricted T-cell activation, we synthesized three altered HA308-317 peptides. The specific binding of altered HA308-317 peptides to HLA-DR1 molecules was examined using flow cytometry. Effects of altered HA308-317 peptides on HLA-DR1-specific T-cell hybridoma were studied by measuring T-cell proliferation and surface expression of CD69 or CD25. The results showed that altered HA308-317 peptides were able to bind to HLA-DR1 molecules and competed with CII263-272 or wildtype HA308-317 peptide. Compared with wildtype CII263-272 or HA308-317, altered HA308-317 peptides did not stimulate significant T-cell proliferation and CD69 or CD25 expression. Furthermore, the altered HA308-317 peptides inhibited HLA-DR1-specific T-cell activation induced by CII263-272 or wildtype HA308-317 peptide, which may suggest an effective therapeutic strategy in inhibition of HLA-DR1-specific T-cell responses in autoimmunity.

Objective: To study the inhibitory role of altered HA308 - 317 peptides in T cell responses in patients with rheumatoid arthritis (RA)., Methods: Peripheral blood mononuclear cells (PBMC) were obtained from 27 HLA-DRB1 positive RA patients. T cell responses to altered HA308-317 peptides were examined by using (3)H incorporation assay. The level of IL-2 and IFNgamma in the supernatants was identified by an enzyme-linked immunosorbent assay. The CD69 expression on T cell surface was studied by using flow cytometry., Results: Compared to wild type CII263 - 272 or HA308 - 317, altered HA308 - 317 peptides failed to stimulate T cell proliferation (P < 0.05) and production of IL-2 as well as IFNgamma and resulted in lower expression of CD69 in RA patients (P < 0.05). SI values for T cell coincubated with altered HA308 - 317 peptides and CII263 - 272 or wild type HA308 - 317 were significantly lower than coincubated with CII263 - 272 and wild type HA308 - 317 alone (P < 0.05)., Conclusion: Substitution of TCR-binding residue of HA308 - 317 yielded weak or non-T-cell stimulating peptides, which might be potentially effective in blocking abnormal T cell responses in RA.

Influenza A virus M-proteins can cause depression in experimental animals 1 day after administration. HA and NA, on the contrary, activate motor activity in animals under the similar conditions. The effect of viral proteins is dose-dependent.

CD4(+)CD25(+) regulatory T cell selection is initiated by high-specificity interactions with self-peptides in the thymus, although how these cells respond to cytokine-derived signals and to re-exposure to self-peptide:MHC complexes in the periphery is not well understood. We have used a transgenic mouse system, in which the peptide that induces thymic selection of a clonal population of CD4(+)CD25(+) regulatory T cells is known, to show that CD4(+)CD25(+) T cells proliferate in response to their selecting self-peptide in vivo. Moreover, they do not proliferate in response to lymphopenia in the absence of the selecting self-peptide, reflecting a low level of expression of the high affinity receptor for IL-7 (CD127) relative to conventional CD4(+) T cells. That their selecting self-peptide is both required for and promotes the peripheral expansion of CD4(+)CD25(+) regulatory T cells may direct their accumulation in sites where the self-peptide is expressed.

Reliable techniques are required to evaluate the plausibility of proposed membrane fusion mechanisms. Here we have studied the kinetics of establishing the lipidic connection between hemagglutinin-expressing cells (HA-cells) and red blood cells (RBC) labeled with octadecylrhodamine, R18, using three different experimental approaches: (1) the most common approach of monitoring the rate of the R18 dequenching in a cuvette with a suspension of RBC/HA-cell complexes; (2) video fluorescence microscopy (VFM) to detect the waiting times before the onset of R18 redistribution, not dequenching, for each RBC attached to an adherent HA-cell; and (3) a new approach based on blockage of RBC fusion to an adherent HA-cell at different time points by lysophosphatidylcholine (LPC), so that only the cell pairs which, at the time of LPC application, had fused or were irreversibly committed to fusion contributed to the final extent of lipid mixing. The LPC blockage and VFM gave very similar estimates for the fusion kinetics, with LPC monitoring also those sites committed to the lipid mixing process. In contrast, R18 dequenching in the cuvette was much slower, i.e., it monitors a much later stage of dye redistribution.

The effects of hemagglutinin (HA) fusion peptide (X-31) on poly(ethylene glycol)- (PEG-) mediated vesicle fusion in three different vesicle systems have been compared: dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV) and palmitoyloleoylphosphatidylcholine (POPC) large unilamellar perturbed vesicles (pert. LUV). POPC LUVs were asymmetrically perturbed by hydrolyzing 2.5% of the outer leaflet lipid with phospholipase A(2) and removing hydrolysis products with BSA. The mixing of vesicle contents showed that these perturbed vesicles fused in the presence of PEG as did DOPC SUV, but unperturbed LUV did not. Fusion peptide had different effects on the fusion of these different types of vesicles: fusion was not induced in the absence of PEG or in unperturbed DOPC LUV even in the presence of PEG. Fusion was enhanced in DOPC SUV at low peptide surface occupancy but hindered at high surface occupancy. Finally, fusion was hindered in proportion to peptide concentration in perturbed POPC LUV. Contents leakage assays demonstrated that the peptide enhanced leakage in all vesicles. The peptide enhanced lipid transfer between both fusogenic and nonfusogenic vesicles. Peptide binding was detected in terms of enhanced tryptophan fluorescence or through transfer of tryptophan excited-state energy to membrane-bound diphenylhexatriene (DPH). The peptide had a higher affinity for vesicles with packing defects (SUV and perturbed LUV). Quasi-elastic light scattering (QELS) indicated that the peptide caused vesicles to aggregate. We conclude that binding of the fusion peptide to vesicle membranes has a significant effect on membrane properties but does not induce fusion. Indeed, the fusion peptide inhibited fusion of perturbed LUV. It can, however, enhance fusion between highly curved membranes that normally fuse when brought into close contact by PEG.

Purpose: Delivery of specific antibodies or immunoglobulin constructs to the respiratory tract may be useful for prophylaxis or active treatment of local or systemic disorders. Therefore, we evaluated the utility of lipid-based hollow-porous microparticles (PulmoSpheres) as a potential delivery vehicle for immunoglobulins., Methods: Lipid-based microparticles loaded with human immunoglobulin (hIgG) or control peptide were synthesized by spray drying and tested for: i) the kinetics of peptide/protein release, using ELISA and bioassays; ii) bioavailability subsequent to nonaqueous liquid instillation into the respiratory tract of BALB/c mice, using ELISA and Western blotting; iii) bioactivity in terms of murine immune response to xenotypic epitopes on human IgG, using ELISA and T cell assays; and iv) mechanisms responsible for the observed enhancement of immune responses, using measurement of antibodies as well as tagged probes., Results: Human IgG and the control peptide were both readily released from the hollow-porous microspheres once added to an aqueous environment, although the kinetics depended on the compound. Nonaqueous liquid instillation of hIgG formulated in PulmoSpheres into the upper and lower respiratory tract of BALB/c mice resulted in systemic biodistribution. The formulated human IgG triggered enhanced local and systemic immune responses against xenotypic epitopes and was associated with receptor-mediated loading of alveolar macrophages., Conclusions: Formulation of immunoglobulins in hollow-porous microparticles is compatible with local and systemic delivery via the respiratory mucosa and may be used as means to trigger or modulate immune responses.

Peptide III is a 20-residue synthetic model peptide based on the fusion peptide of influenza virus A/PR/8/34 strain and takes a secondary structure similar to the original peptide. While conserving the amphiphilic helical nature, 20 peptides to modify the bulkiness of side chains of peptide III were synthesized, and acid-induced membrane destabilization was assessed by aqueous content leakage from large unilamellar vesicles. Substitutions on the hydrophobic side decreased activity but showed less effect on the hydrophilic side, which confirmed the importance of the hydrophobic side for interaction with the membrane. Interestingly, substitution at the 13th Gly residue enhanced the amphiphilic helical nature but severely reduced activity. Correlation between alpha-helical content at acidic pH and the activity was not recognized, suggesting rather that the importance of this site was due to helix termination by glycine which allows N-terminal and C-terminal halves to behave as different secondary structural units.

To better understand the roles of different regions of influenza hemagglutinin in membrane fusion, we have studied the fusion properties of large unilamellar vesicles in the presence of constructs comprising the 127 amino acid ectodomain of the HA2 fragment (FHA2) as well as mutated forms of FHA2 containing single amino acid substitutions, the 95 amino acid truncated form of FHA2 lacking the N-terminal fusion peptide (SHA2), the 20 amino acid N-terminal fusion peptide and the ten amino acid peptide corresponding to the kinked loop region of FHA2. The 100 nm liposomes were made from dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine and cholesterol in equimolar ratio. At pH 5 a high rate of lipid mixing was observed with FHA2 present, even at very low molar concentrations, whereas much lower rates were observed using the shorter constructs: SHA2, the fusion peptide, and the loop peptide. Concentrations of FHA2 which promoted extensive lipid mixing also induced leakage of aqueous contents. Marked effects of FHA2 were also observed with liposomes of egg phosphatidylcholine. All of the changes observed with the liposomes were highly pH-dependent, with only negligible changes occurring at pH 7. The results demonstrate the potent action of FHA2 in promoting lipid mixing and demonstrate the contribution of other regions of the ectodomain of FHA2, in addition to the fusion peptide, to the mechanism of acceleration of membrane fusion. The results also indicate that the pH dependence of fusion is not due solely to changes in the interactions between the HA1 and HA2 subunits. Thus, the "spring loaded energy" is not required to bring about the apposition of the two membranes, considering that FHA2 is already in its thermostable conformation. The acidic amino acid residues in the kinked loop region appear to play a particularly important role in the pH-dependent fusion process as demonstrated by the marked loss of lipid mixing activity of mutant forms of FHA2., (Copyright 1999 Academic Press.)

The mechanism of bilayer unification in biological fusion is unclear. We reversibly arrested hemagglutinin (HA)-mediated cell-cell fusion right before fusion pore opening. A low-pH conformation of HA was required to form this intermediate and to ensure fusion beyond it. We present evidence indicating that outer monolayers of the fusing membranes were merged and continuous in this intermediate, but HA restricted lipid mixing. Depending on the surface density of HA and the membrane lipid composition, this restricted hemifusion intermediate either transformed into a fusion pore or expanded into an unrestricted hemifusion, without pores but with unrestricted lipid mixing. Our results suggest that restriction of lipid flux by a ring of activated HA is necessary for successful fusion, during which a lipidic fusion pore develops in a local and transient hemifusion diaphragm.

Transport from the TGN to the basolateral surface involves a rab/N-ethylmaleimide-sensitive fusion protein (NSF)/soluble NSF attachment protein (SNAP)/SNAP receptor (SNARE) mechanism. Apical transport instead is thought to be mediated by detergent-insoluble sphingolipid-cholesterol rafts. By reducing the cholesterol level of living cells by 60-70% with lovastatin and methyl-beta-cyclodextrin, we show that the TGN-to-surface transport of the apical marker protein influenza virus hemagglutinin was slowed down, whereas the transport of the basolateral marker vesicular stomatitis virus glycoprotein as well as the ER-to-Golgi transport of both membrane proteins was not affected. Reduction of transport of hemagglutinin was accompanied by increased solubility in the detergent Triton X-100 and by significant missorting of hemagglutinin to the basolateral membrane. In addition, depletion of cellular cholesterol by lovastatin and methyl-beta-cyclodextrin led to missorting of the apical secretory glycoprotein gp-80, suggesting that gp-80 uses a raft-dependent mechanism for apical sorting. Our data provide for the first time direct evidence for the functional significance of cholesterol in the sorting of apical membrane proteins as well as of apically secreted glycoproteins.

B- and T-cell responses have been studied after primary and secondary immunizations of mice with iscoms containing influenza virus envelope glycoproteins. After primary immunization both B- and T-cell responses were initiated in the draining lymph nodes. T-cells showed peak activity with respect to proliferation and cytokine production after five to eight days and the highest number of IgG secreting cells (IgG-SC) was recorded at day seven. The responses in the spleen developed slowly but were of longer duration. Cytokines produced by spleen cells included high levels of IFN-gamma and IL-2. After a secondary immunization the frequencies of IgG-SC were drastically increased in both LN and spleen, but decreased rapidly with time. At day eight after the secondary immunization high numbers of IgG-SC were detected in the bone marrow. High titres of IgG1 and IgG2a and substantial titres of IgG2b and IgG3 were detected in serum.

Human polymorphonuclear leukocytes (PMNL) incubated with influenza virus, A/USSR/90/77 (H1N1) or its hemagglutinin produced a significant increase in their PKC activity when compared with untreated PMNL. The activated kinase translocated from cytosol to cellular membrane. The calcium-dependent enzyme activity was inhibited by a specific inhibitor suggesting that alpha and/or beta isoforms of PKC were involved.

Polymyositis is regarded as an autoimmune inflammatory muscle disease. A major subgroup of patients have autoantibodies to cellular histidyl-transfer RNA synthetase (HRS). We have analyzed the role of the autoantigen HRS in the induction of murine myositis in a comparative study of inoculation of BALB/c mice with recombinant HRS protein versus naked DNA coding for HRS. Adult BALB/c mice produced antibodies to human HRS following inoculation with HRS protein and adjuvant, but myositis was not observed. Alternatively, expression plasmid DNA constructs encoding full-length and truncated human HRS were inoculated intramuscularly in gene transfer studies. DNA-inoculated mice produced relatively low anti-HRS antibody titers. However, in contrast to recombinant HRS protein-inoculated mice, HRS gene transfer induced pathology with evidence of cellular infiltration of perivascular and endomysial regions of the inoculated muscle. Multiple inoculations of a plasmid construct encoding a hybrid molecule consisting of HRS and the transferrin receptor cytoplasmic tail induced the highest levels of antibodies and persisting cellular infiltration. Unlike HRS, expression of influenza virus hemagglutinin (HA) following inoculation of an HA plasmid did not induce myositis. Transfer of naked DNA constructs expressing HRS is likely to provide valuable information on the autoimmune response to this protein and its role in the development of myositis.

Antisense oligonucleotides (ODN) were easily introduced into the cytosol of mammalian cells on permeabilization of the plasma membrane by an amphiphilic anionic peptide. The E5CA peptide (GLFEAIAEFIEGGWEGLIEGCA) is an E5 peptide analog derived from the N-terminal segment of the HA2 subunit of influenza virus hemagglutinin. This peptide undergoes a conformational change when the pH shifts from neutral to around 6.0, inducing a transient permeabilization of the plasma membrane. In the presence of the E5CA peptide at pH close to 6.0, fluoresceinylated ODN were rapidly taken up by cells and diffused into the nucleus. The uptake of ODN was dependent on the E5CA peptide concentration and on the duration of the incubation at low pH, as shown by confocal microscopy and flow cytometry analyses. This procedure is suitable for loading adherent cells as well as nonadherent cells with single-stranded or double-stranded ODN. Under optimal conditions, a high percentage of cells were nuclei loaded, and the viability was not affected. This method makes use of a well-defined chemical product without the requirement of any special equipment. It will be useful to study the interactions of single-stranded or double-stranded ODN used as antisense, antigenes, or decoys.

To identify HLA-DR-binding peptides within the human immunodeficiency virus (HIV)-1 proteins. 95 overlapping synthetic peptides representing the entire sequence of gp120-LAI were screened for their capacity to bind to two HLA-DR molecules with distant sequences (DR0401 and DR1101). By using a cell surface competitive binding assay, 56 DR-binding peptides were identified, of which 35 bound to both DR1101 and DR0401. A highly significant concordance was evidenced by statistical analysis between binding of peptides to one and to the other DR molecule, suggesting a high proportion of promiscuity among gp120 peptides, even though no clear sequence pattern accounting for such promiscuity was found. DR-binding peptides were located along the entire gp120 sequence. Yet, the majority of them (42 among 56) were concentrated in seven multiagretopic regions that were arbitrarily defined as regions containing four or more overlapping continuous peptides binding to DR1011 and/or DR0401. A good correlation was found between DR-binding regions or DR-binding peptides defined in this study and promiscuous T helper gp120 epitopes previously described in seropositive individuals. All these results suggest that the identification of multiagretopic DR-binding regions may be a great help for the predicition of protein determinants that have the likelihood of being promiscuous T helper epitopes in humans.

We report that I-Ab-restricted T cell clones, elicited by influenza infection of C57BL/10 mice and specific for the hemagglutinin peptide HA1 186-205, express class II. They respond to peptide stimulation by IL release (IL-3 or IFN-gamma) without a requirement for APC but do not proliferate. Moreover, surface expression of class II requires de novo synthesis in the presence of the stimulatory peptide and is inhibited by coculture with TCR-specific Ab, or brefeldin A or cycloheximide. Clonotypic specificity of peptide induction was confirmed by failure of other allele specific peptides to enhance class II expression. Addition of the viral peptide to T cells induced homotypic adhesion, which provides a physical basis for stabilization of class II-peptide complexes at the cell surface. Extinction of class II expression was evident in the corresponding T cell hybridomas, which might account for the failure to report class II expression by murine T cells. Control studies indicated that class II was not passively acquired from APC by demonstrating 1) failure of processed Ag to induce class II expression, 2) allo-class II (Ak) was not acquired by coculture with peptide and semisyngeneic (H-2 b/k) APC, 3) absence of class II expression by a NP peptide-specific Th2 clone under identical culture conditions, and most significantly, 4) reverse-transcriptase PCR amplification and surface expression of class II using highly purified preparations of FACS-selected CD4+ class II- cells cocultured with the stimulatory peptide.

Interaction of the B cell receptor (BCR) with non-immunogenic receptor ligands can induce a specific state of B cell unresponsiveness as a result of receptor desensitization. Provided it is maintained over time, BCR desensitization may provide the molecular basis for clonal anergy. Using an in vitro model of anti-Ig-mediated BCR desensitization, we assessed the susceptibility of desensitized "anergic" B lymphocytes to activation by B cell superstimulatory influenza virus hemagglutinin (HA). Rabbit anti-mouse Ig antibodies (whole Ig molecule or F(ab')(2)-fragments) totally abolished the response of murine B cells to HA, when added simultaneously with the virus. Pretreatment with the same antibodies, however, yielding a complete unresponsiveness to a subsequent challenge with normally mitogenic anti-Ig reagents, even enhanced the subsequent proliferative response to HA. By contrast, HA-mediated high-rate immunoglobulin synthesis was suppressed after desensitization. BCR-desensitized, HA-stimulated B cells exhibited a hyperexpression of various activation markers (B7, major histocompatibility complex class II, CD25) and served as potent antigen-presenting cells (APC) in a polyclonal model for T lymphocyte activation. These observations suggest a possible scenario for the breaking of natural B cell tolerance, where infections with B cell superstimulatory viruses may lead to the clonal expansion of receptor desensitized, functionally silenced B lymphocytes in vivo.