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.

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.

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.

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.

Neisseria meningitidis type C capsular polysaccharide inhibits lipooligosaccharide-induced cell activation by binding to CD14.

Encapsulated Neisseria meningitidis can invade mucosal barriers and cause systemic diseases. Activation of the innate immune system by conserved meningococcal molecules such as lipooligosaccharides (LOS) is essential for the generation of an effective host immune response. Here we show that the type C capsular polysaccharide of N. meningitidis (MCPS) inhibited LOS-induced interleukin-6 and TNF-alpha secretion from monocytes, and blocked the maturation of dendritic cells induced by LOS, while the capsular polysaccharide from group B streptococcus type III and t(4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll had no such effect. MCPS also inhibited the LOS-induced NF-kappaB activation and phosphorylation of signalling molecules such as ERK1/2, p38 and Jun N-terminal kinase. In a direct binding assay, MCPS manifested a concentration-dependent binding to recombinant lipoprotein binding protein and CD14, the two members of the LOS receptor complex. In addition, the binding of LOS to CD14 and lipopolysaccharide binding protein was inhibited by MCPS. We established that MCPS binding to CD14 is responsible for the inhibition of LOS-mediated cell activation because MCPS inhibition of LOS was reversed when access amounts of CD14 were added to culture media of HEK293 cells expressing TLR4 and MD-2, and the magnitude of recovery in LOS stimulation correlated with the increase in CD14 concentration. These results suggest a new virulence property of meningococcal capsular polysaccharides.

Proteasome inhibition induces TNFR1 shedding from human airway epithelial (NCI-H292) cells.

The type 1 55-kDa TNF receptor (TNFR1) is an important modulator of lung inflammation. Here, we hypothesized that the proteasome might regulate TNFR1 shedding from human airway epithelial cells. Treatment of NCI-H292 human airway epithelial cells for 2 h with the specific proteasome inhibitor clasto-lactacystin beta-lactone induced the shedding of proteolytically cleaved TNFR1 ectodomains. Clasto-lactacystin beta-lactone also induced soluble TNFR1 (sTNFR1) release from the A549 pulmonary epithelial cell line, as well as from primary cultures of human small airway epithelial cells and human umbilical vein endothelial cells. Furthermore, sTNFR1 release induced by clasto-lactacystin beta-lactone was not a consequence of apoptosis or the extracellular release of TNFR1 exosome-like vesicles. The clasto-lactacystin beta-lactone-induced increase in TNFR1 shedding was associated with reductions in cell surface receptors and intracytoplasmic TNFR1 stores that were primarily localized to vesicular structures. As expected, the broad-spectrum zinc metalloprotease inhibitor TNF-alpha protease inhibitor 2 (TAPI-2) attenuated clasto-lactacystin beta-lactone-mediated TNFR1 shedding, which is consistent with its ability to inhibit the zinc metalloprotease-catalyzed cleavage of TNFR1 ectodomains. TAPI-2 also reduced TNFR1 on the cell surface and attenuated the clasto-lactacystin beta-lactone-induced reduction of intracytoplasmic TNFR1 vesicles. This suggests that TNFR1 shedding induced by clasto-lactacystin beta-lactone involves the zinc metalloprotease-dependent trafficking of intracytoplasmic TNFR1 vesicles to the cell surface. Together, these data are consistent with the conclusion that proteasomal activity negatively regulates TNFR1 shedding from human airway epithelial cells, thus identifying previously unrecognized roles for the proteasome and zinc metalloproteases in modulating the generation of sTNFRs.

Release of full-length 55-kDa TNF receptor 1 in exosome-like vesicles: a mechanism for generation of soluble cytokine receptors.

Soluble tumor necrosis factor receptors (TNFRs) are important modulators of TNF bioactivity. Proteolytic cleavage of the 28-kDa ectodomain of TNFR1 has been recognized as the mechanism by which soluble TNFR is shed. We now describe the release of exosome-like vesicles as a mechanism for the generation of soluble, full-length 55-kDa TNFR1. We found unexpectedly that the predominant form of soluble TNFR1 in human serum and lung epithelial lining fluid is a full-length 55-kDa protein. Furthermore, supernatants from human vascular endothelial cells contain only full-length 55-kDa TNFR1 that can be sedimented by high-speed centrifugation, floated on sucrose gradients at a density of 1.1 g/ml, and associated with vesicles that range in diameter from 20 nm to 50 nm. We conclude that the release of TNFR1 exosome-like vesicles represents a previously unrecognized mechanism by which constitutive production of soluble cytokine receptors may be regulated, independent of ectodomain cleavage by receptor sheddases.

Shedding of the type II IL-1 decoy receptor requires a multifunctional aminopeptidase, aminopeptidase regulator of TNF receptor type 1 shedding.

Proteolytic cleavage of the extracellular domain of the type II IL-1 decoy receptor (IL-1RII) generates soluble IL-1-binding proteins that prevent excessive bioactivity by binding free IL-1. In this study we report that an aminopeptidase, aminopeptidase regulator of TNFR1 shedding (ARTS-1), is required for IL-1RII shedding. Coimmunoprecipitation experiments demonstrate an association between endogenous membrane-associated ARTS-1 and a 47-kDa IL-1RII, consistent with ectodomain cleavage of the membrane-bound receptor. A direct correlation exists between ARTS-1 protein expression and IL-1RII shedding, as cell lines overexpressing ARTS-1 have increased IL-1RII shedding and decreased membrane-associated IL-1RII. Basal IL-1RII shedding is absent from ARTS-1 knockout cell lines, demonstrating that ARTS-1 is required for constitutive IL-1RII shedding. Similarly, PMA-mediated IL-1RII shedding is almost entirely ARTS-1-dependent. ARTS-1 expression also enhances ionomycin-induced IL-1RII shedding. ARTS-1 did not alter levels of membrane-associated IL-1RI or IL-1R antagonist release from ARTS-1 cell lines, which suggests that the ability of ARTS-1 to promote shedding of IL-1R family members may be specific for IL-1RII. Further, increased IL-1RII shedding by ARTS-1-overexpressing cells attenuates the biological activity of IL-1beta. We conclude that the ability of ARTS-1 to enhance IL-1RII shedding represents a new mechanism by which IL-1-induced cellular events can be modulated. As ARTS-1 also promotes the shedding of the structurally unrelated 55-kDa, type I TNF receptor and the IL-6R, we propose that ARTS-1 may play an important role in regulating innate immune and inflammatory responses by increasing cytokine receptor shedding.

An aminopeptidase, ARTS-1, is required for interleukin-6 receptor shedding.

Aminopeptidase regulator of TNFR1 shedding (ARTS-1) binds to the type I tumor necrosis factor receptor (TNFR1) and promotes receptor shedding. Because hydroxamic acid-based metalloprotease inhibitors prevent shedding of both TNFR1 and the interleukin-6 receptor (IL-6Ralpha), we hypothesized that ARTS-1 might also regulate shedding of IL-6Ralpha, a member of the type I cytokine receptor superfamily that is structurally different from TNFR1. Reciprocal co-immunoprecipitation experiments identified that membrane-associated ARTS-1 directly binds to a 55-kDa IL-6Ralpha, a size consistent with soluble IL-6Ralpha generated by ectodomain cleavage of the membrane-bound receptor. Furthermore, ARTS-1 promoted IL-6Ralpha shedding, as demonstrated by a direct correlation between increased membrane-associated ARTS-1 protein, increased IL-6Ralpha shedding, and decreased membrane-associated IL-6Ralpha in cell lines overexpressing ARTS-1. The absence of basal IL-6Ralpha shedding from arts-1 knock-out cells identified that ARTS-1 was required for constitutive IL-6Ralpha shedding. Furthermore, the mechanism of constitutive IL-6Ralpha shedding requires ARTS-1 catalytic activity. Thus, ARTS-1 promotes the shedding of two cytokine receptor superfamilies, the type I cytokine receptor superfamily (IL-6Ralpha) and the TNF receptor superfamily (TNFR1). We propose that ARTS-1 is a multifunctional aminopeptidase that may modulate inflammatory events by promoting IL-6Ralpha and TNFR1 shedding.

Identification of ARTS-1 as a novel TNFR1-binding protein that promotes TNFR1 ectodomain shedding.

Proteolytic cleavage of TNF receptor 1 (TNFR1) generates soluble receptors that regulate TNF bioactivity. We hypothesized that the mechanism of TNFR1 shedding might involve interactions with regulatory ectoproteins. Using a yeast two-hybrid approach, we identified ARTS-1 (aminopeptidase regulator of TNFR1 shedding) as a type II integral membrane protein that binds to the TNFR1 extracellular domain. In vivo binding of membrane-associated ARTS-1 to TNFR1 was confirmed by coimmunoprecipitation experiments using human pulmonary epithelial and umbilical vein endothelial cells. A direct relationship exists between membrane-associated ARTS-1 protein levels and concordant changes in TNFR1 shedding. Cells overexpressing ARTS-1 demonstrated increased TNFR1 shedding and decreased membrane-associated TNFR1, while cells expressing antisense ARTS-1 mRNA demonstrated decreased membrane-associated ARTS-1, decreased TNFR1 shedding, and increased membrane-associated TNFR1. ARTS-1 neither bound to TNFR2 nor altered its shedding, suggesting specificity for TNFR1. Although a recombinant ARTS-1 protein demonstrated selective aminopeptidase activity toward nonpolar amino acids, multiple lines of negative evidence suggest that ARTS-1 does not possess TNFR1 sheddase activity. These data indicate that ARTS-1 is a multifunctional ectoprotein capable of binding to and promoting TNFR1 shedding. We propose that formation of a TNFR1-ARTS-1 molecular complex represents a novel mechanism by which TNFR1 shedding is regulated.