Epstein Barr Virus: Development of Vaccines and Immune Cell Therapy for EBV-Associated Diseases.

Epstein-Barr virus (EBV) is the first human tumor virus discovered and is strongly implicated in the etiology of multiple lymphoid and epithelial cancers. Each year EBV associated cancers account for over 200,000 new cases of cancer and cause 150,000 deaths world-wide. EBV is also the primary cause of infectious mononucleosis, and up to 70% of adolescents and young adults in developed countries suffer from infectious mononucleosis. In addition, EBV has been shown to play a critical role in the pathogenesis of multiple sclerosis. An EBV prophylactic vaccine that induces neutralizing antibodies holds great promise for prevention of EBV associated diseases. EBV envelope proteins including gH/gL, gB and gp350 play key roles in EBV entry and infection of target cells, and neutralizing antibodies elicited by each of these proteins have shown to prevent EBV infection of target cells and markedly decrease EBV titers in the peripheral blood of humanized mice challenged with lethal dose EBV. Recent studies demonstrated that immunization with the combination of gH/gL, gB and/or gp350 induced markedly increased synergistic EBV neutralizing activity compared to immunization with individual proteins. As previous clinical trials focused on gp350 alone were partially successful, the inclusion of gH/gL and gB in a vaccine formulation with gp350 represents a promising approach of EBV prophylactic vaccine development. Therapeutic EBV vaccines have also been tested clinically with encouraging results. Immunization with various vaccine platforms expressing the EBV latent proteins EBNA1, LMP1, and/or LMP2 promoted specific CD4+ and CD8+ cytotoxic responses with anti-tumor activity. The addition of EBV envelope proteins gH/gL, gB and gp350 has the potential to increase the efficacy of a therapeutic EBV vaccine. The immune system plays a critical role in the control of tumors, and immune cell therapy has emerged as a promising treatment of cancers. Adoptive T-cell therapy has been successfully used in the prevention and treatment of post-transplant lymphoproliferative disorder. Chimeric antigen receptor T cell therapy and T cell receptor engineered T cell therapy targeting EBV latent proteins LMP1, LMP2 and/or EBNA1 have been in development, with the goal to increase the specificity and efficacy of treatment of EBV associated cancers.

Immunization with Epstein-Barr Virus Core Fusion Machinery Envelope Proteins Elicit High Titers of Neutralizing Activities and Protect Humanized Mice from Lethal Dose EBV Challenge.

Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and is strongly implicated in the etiology of multiple lymphoid and epithelial cancers. EBV core fusion machinery envelope proteins gH/gL and gB coordinately mediate EBV fusion and entry into its target cells, B lymphocytes and epithelial cells, suggesting these proteins could induce antibodies that prevent EBV infection. We previously reported that the immunization of rabbits with recombinant EBV gH/gL or trimeric gB each induced markedly higher serum EBV-neutralizing titers for B lymphocytes than that of the leading EBV vaccine candidate gp350. In this study, we demonstrated that immunization of rabbits with EBV core fusion machinery proteins induced high titer EBV neutralizing antibodies for both B lymphocytes and epithelial cells, and EBV gH/gL in combination with EBV trimeric gB elicited strong synergistic EBV neutralizing activities. Furthermore, the immune sera from rabbits immunized with EBV gH/gL or trimeric gB demonstrated strong passive immune protection of humanized mice from lethal dose EBV challenge, partially or completely prevented death respectively, and markedly decreased the EBV load in peripheral blood of humanized mice. These data strongly suggest the combination of EBV core fusion machinery envelope proteins gH/gL and trimeric gB is a promising EBV prophylactic vaccine.

Immunization with Human Cytomegalovirus Core Fusion Machinery and Accessory Envelope Proteins Elicit Strong Synergistic Neutralizing Activities.

Human cytomegalovirus (HCMV) core fusion machinery proteins gB and gH/gL, and accessory proteins UL128/UL130/UL131A, are the key envelope proteins that mediate HCMV entry into and infection of host cells. To determine whether these HCMV envelope proteins could elicit neutralizing activities synergistically, we immunized rabbits with individual or various combinations of these proteins adsorbed to aluminum hydroxide mixed with CpG-ODN. We then analyzed serum neutralizing activities with multiple HCMV laboratory strains and clinical isolates. HCMV trimeric gB and gH/gL elicited high and moderate titers of HCMV neutralizing activity, respectively. HCMV gB in combination with gH/gL elicited up to 17-fold higher HCMV neutralizing activities compared to the sum of neutralizing activity elicited by the individual proteins analyzed with both fibroblasts and epithelial cells. HCMV gB+gH/gL+UL128/UL130/UL131A in combination increased the neutralizing activity up to 32-fold compared to the sum of neutralizing activities elicited by the individual proteins analyzed with epithelial cells. Adding UL128/UL130/UL131A to gB and gH/gL combination did not increase further the HCMV neutralizing activity analyzed with fibroblasts. These data suggest that the combination of HCMV core fusion machinery envelope proteins gB+gH/gL or the combination of gB and pentameric complex could be ideal vaccine candidates that would induce optimal immune responses against HCMV infection.

Immunization of Rabbits with Recombinant Human Cytomegalovirus Trimeric versus Monomeric gH/gL Protein Elicits Markedly Higher Titers of Antibody and Neutralization Activity.

Congenital human cytomegalovirus (HCMV) infection and HCMV infection of immunosuppressed patients cause significant morbidity and mortality, and vaccine development against HCMV is a major public health priority. HCMV envelope glycoproteins gB, gH, and gL, which constitute the core fusion machinery, play critical roles in HCMV fusion and entry into host cells. HCMV gB and gH/gL have been reported to elicit potent neutralizing antibodies. Recently, the gB/gH/gL complex was identified in the envelope of HCMV virions, and 16-50% of the total gH/gL bound to gB, forming the gB/gH/gL complex. These findings make the gB/gH/gL a unique HCMV vaccine candidate. We previously reported the production of HCMV trimeric gB and gH/gL heterodimers, and immunization with a combination of trimeric gB and gH/gL heterodimers elicited strong synergistic HCMV-neutralizing activity. To further improve the immunogenicity of gH/gL, we produced trimeric gH/gL. Rabbits immunized with HCMV trimeric gH/gL induced up to 38-fold higher serum titers of gH/gL-specific IgG relative to HCMV monomeric gH/gL, and elicited ~10-fold higher titers of complement-dependent and complement-independent HCMV-neutralizing activity for both epithelial cells and fibroblasts. HCMV trimeric gH/gL in combination with HCMV trimeric gB would be a novel promising HCMV vaccine candidate that could induce highly potent neutralizing activities.

Development of novel vaccines against human cytomegalovirus.

Congenital human cytomegalovirus (HCMV) infection and HCMV infection of the immunosuppressed patients cause significant morbidity and mortality, and vaccine development against HCMV is a major public health priority. Efforts to develop HCMV vaccines have been ongoing for 50 y, though no HCMV vaccine has been licensed; encouraging and promising results have obtained from both preclinical and clinical trials. HCMV infection induces a wide range of humoral and T cell-mediated immune responses, and both branches of immunity are correlated with protection. In recent years, there have been novel approaches toward the development of HCMV vaccines and demonstrated that vaccine candidates could potentially provide superior protection over natural immunity acquired following HCMV infection. Further, rationally designed HCMV protein antigens that express native conformational epitopes could elicit optimal immune response. HCMV vaccine candidates, using a multi-antigen approach, to maximize the elicited protective immunity will most likely be successful in development of HCMV vaccine.

Decreased nematode clearance and anti-phosphorylcholine-specific IgM responses in mannose-binding lectin-deficient mice.

Brugia malayi is a nematode that causes human lymphatic filariasis. Previously, we showed that mannose-binding lectin (MBL)-A is necessary for clearance of B. malayi microfilariae in mice and presence of MBL-A is linked with maximal levels of parasite-specific IgM. Common human MBL gene polymorphisms result in low MBL expression and lead to recurring bacterial infections. Furthermore, these low-expressing human MBL polymorphisms result in greatly increased susceptibility to lymphatic filarial infection. Indeed, gain of new filarial infections over a 30-year period are 10-fold higher in people with low, compared to high, MBL-expression phenotypes. Human MBL closely resembles mouse MBL-C, rather than MBL-A; therefore, we examined the role of mouse MBL-C in clearance of microfilariae. Absence of MBL-C alone, or both MBL-A and -C, resulted in delayed clearance of microfilariae and reduced parasite-specific IgM in mice. There were few profound changes in B cell sub-populations or in the ability of MBL-deficient mice to respond to T-dependent or T-independent antigens. However, absence of MBL-A and/or MBL-C resulted in reduced IgM to phosphorylcholine, a constituent of filarial and bacterial antigens, suggesting that inability to form proficient antibody responses to this moiety leads to lack of microfilarial clearance and overall susceptibility to filariasis.

Novel trimeric human cytomegalovirus glycoprotein B elicits a high-titer neutralizing antibody response.

Human cytomegalovirus (HCMV) is a major cause of disability in congenitally infected infants and in the immunosuppressed. There is currently no licensed prophylactic HCMV vaccine. The HCMV envelope glycoprotein B (gB) is considered a major vaccine target antigen based on its critical role in mediating viral-host cell fusion and thus viral entry. The natural conformation of HCMV gB within the viral envelope is a trimer, but there has been no reported success in producing a recombinant trimeric gB suitable for vaccine use. Phase II clinical trials of a monomeric recombinant gB protein demonstrated 50% efficacy in preventing HCMV infection in seronegative women of reproductive age, and in reducing viremia in solid organ transplantation recipients. We now report the production of a uniformly trimeric recombinant HCMV gB protein in Chinese ovary cells, as demonstrated by Western blot analysis under modified non-reducing conditions and size exclusion chromatography with multi-angle scattering. Immunization of mice with trimeric HCMV gB induced up to 11-fold higher serum titers of total gB-specific IgG relative to monomeric HCMV gB using Alum + CpG as adjuvants. Further, trimeric HCMV gB elicited 50-fold higher complement-independent and 20-fold higher complement-dependent HCMV neutralizing titers compared to monomeric HCMV gB using the fibroblast cell line, MRC-5, and up to 6-fold higher complement-independent and -dependent HCMV neutralizing titers using the epithelial cell line, ARPE-19. The markedly enhanced HCMV neutralizing activity in response to trimeric HCMV gB was also observed using an additional four distinct clinical HCMV isolates. These data support a role for trimeric HCMV gB as an important component for clinical testing of a prophylactic HCMV vaccine.

Distinct Immunologic Properties of Soluble Versus Particulate Antigens.

Antigens in particulate form have distinct immunologic properties relative to soluble antigens. An understanding of the mechanisms and functional consequences of the distinct immunologic pathways engaged by these different forms of antigen is particularly relevant to the design of vaccines. It is also relevant regarding the use of therapeutic human proteins in clinical medicine that have been shown to aggregate, and perhaps as a result, elicit autoantibodies.

Rabbits immunized with Epstein-Barr virus gH/gL or gB recombinant proteins elicit higher serum virus neutralizing activity than gp350.

Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and has been strongly implicated in the etiology of multiple epithelial and lymphoid cancers, such as nasopharyngeal carcinoma, gastric carcinoma, Hodgkin lymphoma, Burkitt lymphoma, non-Hodgkin lymphoma and post-transplant lymphoproliferative disorder. There is currently no licensed prophylactic vaccine for EBV. Most efforts to develop prophylactic vaccines have focused on EBV gp350, which binds to CD21/CD35 to gain entry into B cells, and is a major target of serum neutralizing antibody in EBV seropositive humans. However, a recombinant monomeric gp350 protein failed to prevent EBV infection in a phase II clinical trial. Thus, alternative or additional target antigens may be necessary for a successful prophylactic vaccine. EBV gH/gL and gB proteins coordinately mediate EBV fusion and entry into B cells and epithelial cells, strongly suggesting that vaccination with these proteins might elicit antibodies that will prevent EBV infection. We produced recombinant trimeric and monomeric EBV gH/gL heterodimeric proteins and a trimeric EBV gB protein, in addition to tetrameric and monomeric gp350(1-470) proteins, in Chinese hamster ovary cells. We demonstrated that vaccination of rabbits with trimeric and monomeric gH/gL, trimeric gB, and tetrameric gp350(1-470) induced serum EBV-neutralizing titers, using cultured human B cells, that were >100-fold, 20-fold, 18-fold, and 4-fold higher, respectively, than monomeric gp350(1-470). These data strongly suggest a role for testing EBV gH/gL and EBV gB in a future prophylactic vaccine to prevent EBV infection of B cells, as well as epithelial cells.

Differential regulation of polysaccharide-specific antibody responses to isolated polysaccharides, conjugate vaccines, and intact Gram-positive versus Gram-negative extracellular bacteria.

Bacterial capsular polysaccharides are major virulence factors and are key targets in a number of licensed anti-bacterial vaccines. Their major characteristics are their large molecular weight and expression of repeating antigenic epitopes that mediate multivalent B cell receptor cross-linking. In addition, since the majority of these antigens cannot associate with MHC-II they fail to recruit CD4+ T cell help and are referred to as T cell-independent antigens. In this review I will discuss a series of studies from my laboratory that have underscored the importance of understanding polysaccharide-specific antibody responses within the context in which the PS is expressed (i.e. in isolation, as a component of conjugate vaccines, and expressed naturally by intact bacteria). We have shown that multivalent B cell receptor crosslinking, as mediated by polysaccharides, uniquely determines the qualitative response of the B cell to subsequent stimuli, but by itself is insufficient to induce antibody secretion or class switching. For these latter events to occur, second signals must act in concert with primary signals derived from the B cell receptor. The co-expression of polysaccharide and protein within intact bacteria promotes recruitment of CD4+ T cell help for the associated PS-specific IgG response, in contrast to isolated polysaccharides. Further, the particulate nature of extracellular bacteria confers properties to the polysaccharide-specific IgG response that makes it distinct immunologically from soluble conjugate vaccines. Finally, the underlying biochemical and/or structural differences that distinguish Gram-positive and Gram-negative bacteria appear to play critical roles in differentially regulating the associated polysaccharide-specific IgG responses to these groups of pathogens. These studies have a number of implications for the understanding and future design of polysaccharide-based vaccines.

Distinct Mechanisms Underlie Boosted Polysaccharide-Specific IgG Responses Following Secondary Challenge with Intact Gram-Negative versus Gram-Positive Extracellular Bacteria.

Priming of mice with intact, heat-killed cells of Gram-negative Neisseria meningitidis, capsular serogroup C (MenC) or Gram-positive group B Streptococcus, capsular type III (GBS-III) bacteria resulted in augmented serum polysaccharide (PS)-specific IgG titers following booster immunization. Induction of memory required CD4(+) T cells during primary immunization. We determined whether PS-specific memory for IgG production was contained within the B cell and/or T cell populations, and whether augmented IgG responses following booster immunization were also dependent on CD4(+) T cells. Adoptive transfer of purified B cells from MenC- or GBS-III-primed, but not naive mice resulted in augmented PS-specific IgG responses following booster immunization. Similar responses were observed when cotransferred CD4(+) T cells were from primed or naive mice. Similarly, primary immunization with unencapsulated MenC or GBS-III, to potentially prime CD4(+) T cells, failed to enhance PS-specific IgG responses following booster immunization with their encapsulated isogenic partners. Furthermore, in contrast to GBS-III, depletion of CD4(+) T cells during secondary immunization with MenC or another Gram-negative bacteria, Acinetobacter baumannii, did not inhibit augmented PS-specific IgG booster responses of mice primed with heat-killed cells. Also, in contrast with GBS-III, booster immunization of MenC-primed mice with isolated MenC-PS, a TI Ag, or a conjugate of MenC-PS and tetanus toxoid elicited an augmented PS-specific IgG response similar to booster immunization with intact MenC. These data demonstrate that memory for augmented PS-specific IgG booster responses to Gram-negative and Gram-positive bacteria is contained solely within the B cell compartment, with a differential requirement for CD4(+) T cells for augmented IgG responses following booster immunization.

Distinct Cellular Pathways for Induction of CD4+ T Cell-Dependent Antibody Responses to Antigen Expressed by Intact Bacteria Versus Isolated Soluble Antigen.

Uptake of intact bacteria and soluble Ags by APCs is mediated by phagocytosis and endocytosis or pinocytosis, respectively. Thus, we predicted that injection of clodronate-containing liposomes (CLs), which selectively deplete cells efficient in phagocytosis, would inhibit murine CD4(+) T cell-dependent IgG responses to Ags expressed by intact bacteria but not isolated soluble Ags. Surprisingly, injection of CLs markedly inhibited protein-specific IgG responses to intact, heat-killed Streptococcus pneumoniae, as well as a soluble OVA-polysaccharide conjugate or OVA alone. IgG anti-polysaccharide responses to bacteria and conjugate were also reduced, but more modestly. In both instances, CL-mediated inhibition was associated with a significant reduction in induced germinal centers and CD4(+) germinal center T follicular helper cells. However, CL injection, which largely abrogated the proliferative response of adoptively transferred OVA peptide-specific-transgenic CD4(+) T cells in response to immunization with S. pneumoniae expressing OVA peptide, did not inhibit T cell proliferation in response to OVA-polysaccharide conjugate or OVA. In this regard, monocyte-derived cells, depleted by CLs, internalized S. pneumoniae in vivo, whereas CD11c(low) dendritic cells, unaffected by CL injection, internalized soluble OVA. Ex vivo isolation and coculture of these respective APCs from S. pneumoniae- or OVA-immunized mice with OVA-specific T cells, in the absence of exogenous Ag, demonstrated their selective ability to induce T cell activation. These data suggest that, although distinct APCs initiate CD4(+) T cell activation in response to Ag expressed by intact bacteria versus Ag in soluble form, CL-sensitive cells appear to be necessary for the subsequent IgG responses to both forms of Ag.

Pneumococcal Surface Protein A Plays a Major Role in Streptococcus pneumoniae-Induced Immunosuppression.

Intact, inactivated Streptococcus pneumoniae [including the unencapsulated S. pneumoniae, serotype 2 strain (R36A)] markedly inhibits the humoral immune response to coimmunized heterologous proteins, a property not observed with several other intact Gram-positive or Gram-negative bacteria. In this study, we determined the nature of this immunosuppressive property. Because phosphorylcholine (PC), a major haptenic component of teichoic acid in the S. pneumoniae cell wall, and lipoteichoic acid in the S. pneumoniae membrane were previously reported to be immunosuppressive when derived from filarial parasites, we determined whether R36A lacking PC (R36A(pc-)) was inhibitory. Indeed, although R36A(pc-) exhibited a markedly reduced level of inhibition of the IgG response to coimmunized chicken OVA (cOVA), no inhibition was observed when using several other distinct PC-expressing bacteria or a soluble, protein-PC conjugate. Further, treatment of R36A with periodate, which selectively destroys PC residues, had no effect on R36A-mediated inhibition. Because R36A(pc-) also lacks choline-binding proteins (CBPs) that require PC for cell wall attachment, and because treatment of R36A with trypsin eliminated its inhibitory activity, we incubated R36A in choline chloride, which selectively strips CBPs from its surface. R36A lacking CBPs lost most of its inhibitory property, whereas the supernatant of choline chloride-treated R36A, containing CBPs, was markedly inhibitory. Coimmunization studies using cOVA and various S. pneumoniae mutants, each genetically deficient in one of the CBPs, demonstrated that only S. pneumoniae lacking the CBP pneumococcal surface protein A lost its ability to inhibit the IgG anti-cOVA response. These results strongly suggest that PspA plays a major role in mediating the immunosuppressive property of S. pneumoniae.

Autologous albumin enhances the humoral immune response to capsular polysaccharide covalently coattached to bacteria-sized latex beads.

Abundant autologous proteins, like serum albumin, should be immunologically inert. However, individuals with no apparent predisposition to autoimmune disease can develop immune responses to autologous therapeutic proteins. Protein aggregation is a potential major trigger of these responses. Adsorption of proteins to particles provides macromolecular size and may generate structural changes in the protein, resembling aggregation. Using aldehyde/sulfate latex beads coated with murine serum albumin (MSA), we found that BALB/c mice mounted MSA-specific IgG responses that were dependent on CD4(+) T cells. IgGs were specific for MSA adsorbed to solid surfaces and noncross-reactive with human, bovine, or pig albumins. T cells induced in response to MSA augmented the primary and induced boosted secondary IgG and IgM responses specific for the T cell-independent antigen, capsular polysaccharide of Streptococcus pneumoniae type 14 (PPS14), when the latter was attached to the same bead. Similar to the anti-MSA IgG response, the boosted PPS14-specific IgG secondary response was CD4(+) T-cell dependent, displayed a typical carrier effect, and was enhanced by, but did not require, Toll-like receptor stimulation. These results provide a potential mechanism for the induction of responses to autoantigens unable to induce specific T-cell responses, and provide new insights into polysaccharide-specific immunity.

Noncovalent association of protein and capsular polysaccharide on bacteria-sized latex beads as a model for polysaccharide-specific humoral immunity to intact gram-positive extracellular bacteria.

Intact Streptococcus pneumoniae expressing type 14 capsular polysaccharide (PPS14) and type III S. agalactiae containing a PPS14 core capsule identical to PPS14 exhibit noncovalent associations of PPS14 and bacterial protein, in contrast to soluble covalent conjugates of these respective Ags. Both bacteria and conjugates induce murine PPS14-specific IgG responses dependent on CD4⁺ T cells. Further, secondary immunization with conjugate and S. agalactiae, although not S. pneumoniae, results in a boosted response. However, in contrast to conjugate, PPS14-specific IgG responses to bacteria lack affinity maturation use the 44.1-idiotype and are dependent on marginal zone B cells. To better understand the mechanism underlying this dichotomy, we developed a minimal model of intact bacteria in which PPS14 and pneumococcal surface protein A (PspA) were stably attached to 1 µm (bacteria-sized) latex beads, but not directly linked to each other, in contrast to PPS14-PspA conjugate. Beads coated simultaneously with PPS14+[PspA], similar to conjugate, induced in mice boosted PPS14-specific IgG secondary responses, dependent on T cells and ICOS-dependent costimulation, and in which priming could be achieved with PspA alone. In contrast to conjugate, but similar to intact bacteria, the primary PPS14-specific IgG response to beads coated simultaneously with PPS14+[PspA] peaked rapidly, with the secondary response highly enriched for the 44.1-idiotype and lacking affinity maturation. These results demonstrate that noncovalent association in a particle, of polysaccharide and protein, recapitulates essential immunologic characteristics of intact bacteria that are distinct from soluble covalent conjugates of these respective Ags.

Immunosuppressive property within the Streptococcus pneumoniae cell wall that inhibits generation of T follicular helper, germinal center, and plasma cell response to a coimmunized heterologous protein.

We previously demonstrated that intact, inactivated Streptococcus pneumoniae (unencapsulated strain R36A) inhibits IgG responses to a number of coimmunized soluble antigens (Ags). In this study, we investigated the mechanism of this inhibition and whether other extracellular bacteria exhibited similar effects. No inhibition was observed if R36A was given 24 h before or after immunization with soluble chicken ovalbumin (cOVA), indicating that R36A acts transiently during the initiation of the immune response. Using transgenic cOVA-specific CD4(+) T cells, we observed that R36A had no significant effect on T-cell activation (24 h) or generation of regulatory T cells (day 7) and only a modest effect on T-cell proliferation (48 to 96 h) in response to cOVA. However, R36A mediated a significant reduction in the formation of Ag-specific splenic germinal center T follicular helper (GC Tfh) and GC B cells and antibody-secreting cells in the spleen and bone marrow in response to cOVA or cOVA conjugated to 4-hydroxy-3-nitrophenylacetyl hapten (NP-cOVA). Of note, the inhibitory effect of intact R36A on the IgG anti-cOVA response could be reproduced using R36A-derived cell walls. In contrast to R36A, neither inactivated, unencapsulated, intact Neisseria meningitidis nor Streptococcus agalactiae inhibited the OVA-specific IgG response. These results suggest a novel immunosuppressive property within the cell wall of Streptococcus pneumoniae.

B cell-specific deficiencies in mTOR limit humoral immune responses.

Generation of high-affinity Abs in response to Ags/infectious agents is essential for developing long-lasting immune responses. B cell maturation and Ab responses to Ag stimulation require Ig somatic hypermutation (SHM) and class-switch recombination (CSR) for high-affinity responses. Upon immunization with either the model Ag 4-hydroxy-3-nitrophenylacetyl hapten (NP) conjugated to chicken γ globulin lysine (NP-CGG) or heat-killed Streptococcus pneumoniae capsular type 14 protein (Pn14), knock-in (KI) mice hypomorphic for mTOR function had a decreased ability to form germinal centers, develop high-affinity anti-NP-specific or anti-Pn14-specific Abs, and perform SHM/CSR. Hypomorphic mTOR mice also had a high mortality (40%) compared with wild-type (WT) (0%) littermates and had lower pneumococcal surface protein A-specific Ab titers when immunized and challenged with live S. pneumoniae infection. Mice with mTOR deleted in their B cell lineage (knockout [KO]) also produced fewer splenic germinal centers and decreased high-affinity Ab responses to NP-CGG than did their WT littermates. CSR rates were lower in mTOR KI and KO mice, and pharmacologic inhibition of mTOR in WT B cells resulted in decreased rates of ex vivo CSR. RNA and protein levels of activation-induced cytidine deaminase (AID), a protein essential for SHM and CSR, were lower in B cells from both KI and B cell-specific KO mice, concomitant with increases in phosphorylated AKT and FOXO1. Rescue experiments increasing AID expression in KI B cells restored CSR levels to those in WT B cells. Thus, mTOR plays an important immunoregulatory role in the germinal center, at least partially through AID signaling, in generating high-affinity Abs.

A novel tetrameric gp350 1-470 as a potential Epstein-Barr virus vaccine.

Infectious mononucleosis and B-cell transformation in response to infection with Epstein-Barr virus (EBV) is dependent upon binding of the EBV envelope glycoprotein gp350 to CD21 on B-cells. Gp350-specific antibody comprises most of the EBV neutralizing activity in the serum of infected patients, making this protein a promising target antigen for a prophylactic EBV vaccine. We describe a novel, tetrameric gp350-based vaccine that exhibits markedly enhanced immunogenicity relative to its monomeric counterpart. Plasmid DNA was constructed for synthesis, within transfected CHO cells, of a tetrameric, truncated (a.a. 1-470) gp350 protein (gp350(1-470)). Tetrameric gp350(1-470) induced ≈ 20-fold higher serum titers of gp350(1-470)-specific IgG and >19-fold enhancements in neutralizing titers at the highest dose, and was >25-fold more immunogenic on a per-weight basis than monomeric gp350(1-470). Further, epidermal immunization with plasmid DNA encoding gp350(1-470) tetramer induced 8-fold higher serum titers of gp350(1-470)-specific IgG relative to monomer. Tetrameric gp350(1-470) binding to human CD21 was >24-fold more efficient on a per-weight basis than monomer, but neither tetramer nor monomer mediated polyclonal human B-cell activation. Finally, the introduction of strong, universal tetanus toxoid (TT)-specific CD4+ T-cell epitopes into the tetrameric gp350(1-470) had no effect on the gp350(1-470)-specific IgG response in naïve mice, and resulted in suppressed gp350(1-470)-specific IgG responses in TT-primed mice. Collectively, these data suggest that tetrameric gp350(1-470) is a potentially promising candidate for testing as a prophylactic EBV vaccine, and that protein multimerization, using the approach described herein, is likely to be clinically relevant for enhancing the immunogenicity of other proteins of vaccine interest.

Novel synthetic (poly)glycerolphosphate-based antistaphylococcal conjugate vaccine.

Staphylococcal infections are a major source of global morbidity and mortality. Currently there exists no antistaphylococcal vaccine in clinical use. Previous animal studies suggested a possible role for purified lipoteichoic acid as a vaccine target for eliciting protective IgG to several Gram-positive pathogens. Since the highly conserved (poly)glycerolphosphate backbone of lipoteichoic acid is a major antigenic target of the humoral immune system during staphylococcal infections, we developed a synthetic method for producing glycerol phosphoramidites to create a covalent 10-mer of (poly)glycerolphosphate for potential use in a conjugate vaccine. We initially demonstrated that intact Staphylococcus aureus elicits murine CD4(+) T cell-dependent (poly)glycerolphosphate-specific IgM and IgG responses in vivo. Naive mice immunized with a covalent conjugate of (poly)glycerolphosphate and tetanus toxoid in alum plus CpG-oligodeoxynucleotides produced high secondary titers of serum (poly)glycerolphosphate-specific IgG. Sera from immunized mice enhanced opsonophagocytic killing of live Staphylococcus aureus in vitro. Mice actively immunized with the (poly)glycerolphosphate conjugate vaccine showed rapid clearance of staphylococcal bacteremia in vivo relative to mice similarly immunized with an irrelevant conjugate vaccine. In contrast to purified, natural lipoteichoic acid, the (poly)glycerolphosphate conjugate vaccine itself exhibited no detectable inflammatory activity. These data suggest that a synthetic (poly)glycerolphosphate-based conjugate vaccine will contribute to active protection against extracellular Gram-positive pathogens expressing this highly conserved backbone structure in their membrane-associated lipoteichoic acid.

Inflammatory monocytes are critical for induction of a polysaccharide-specific antibody response to an intact bacterium.

Although inflammatory monocytes (IM) (CD11b(+)Ly6C(hi) cells) have been shown to play important roles in cell-mediated host protection against intracellular bacteria, protozoans, and fungi, their potential impact on humoral immune responses to extracellular bacteria are unknown. IM, localized largely to the splenic marginal zone of naive CD11b-diphtheria toxin (DT) receptor bone marrow-chimeric mice were selectively depleted following treatment with DT, including no reduction of CD11b(+) peritoneal B cells. Depletion of IM resulted in a marked reduction in the polysaccharide (PS)-specific, T cell-independent IgM, and T cell-dependent IgG responses to intact, heat-killed Streptococcus pneumoniae with no effect on the associated S. pneumoniae protein-specific IgG response or on the PS- and protein-specific IgG responses to a soluble pneumococcal conjugate vaccine. IM acted largely within the first 48 h following the initiation of the immune response to S. pneumoniae to induce the subsequent production of PS-specific IgM and IgG. Adoptive transfer of highly purified IM from wild-type mice into DT-treated CD11b-DT receptor mice completely restored the defective PS-specific Ig response to S. pneumoniae. IM were phenotypically and functionally distinct from circulating CD11b(+)CD11c(low)Ly6G/C cells (immature blood dendritic cells), previously described to play a role in Ig responses to S. pneumoniae, in that they were CD11c(-) as well as Ly6C(hi) and did not internalize injected S. pneumoniae during the early phase of the response. These data are the first, to our knowledge, to establish a critical role for IM in the induction of an Ig response to an intact extracellular bacterium.