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Background: Current influenza vaccines deliver satisfactory results in young people but are less effective in the elderly. Development of vaccines for an ever-increasing aging population has been an arduous challenge due to immunosenescence that impairs the immune response in the aged, both quantitatively and qualitatively., Results: To potentially enhance vaccine efficacy in the elderly, we investigated the immunogenicity and cross-protection of influenza hemagglutinin virus-like particles (HA-VLP) incorporated with glycosylphosphatidylinositol (GPI)-anchored cytokine-adjuvants (GPI-GM-CSF and GPI-IL-12) via protein transfer in aged mice. Lung viral replication against homologous and heterologous influenza viruses was significantly reduced in aged mice after vaccination with cytokine incorporated VLPs (HA-VLP-Cyt) in comparison to HA-VLP alone. Enhanced IFN-γ + CD4 + and IFN-γ + CD8 + T cell responses were also observed in aged mice immunized with HA-VLP-Cyt when compared to HA-VLP alone., Conclusions: Cytokine-adjuvanted influenza HA-VLP vaccine induced enhanced protective response against homologous influenza A virus infection in aged mice. Influenza HA-VLP vaccine with GPI-cytokines also induced enhanced T cell responses correlating with better protection against heterologous infection in the absence of neutralizing antibodies. The results suggest that a vaccination strategy using cytokine-adjuvanted influenza HA-VLPs could be used to enhance protection against influenza A virus in the elderly., (© 2023. The Author(s).)

Several approaches have produced an effective vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since millions of people are exposed to influenza virus and SARS-CoV-2, it is of great interest to develop a two-in-one vaccine that will be able to protect against infection of both viruses. We have developed a hybrid vaccine for SARS-CoV-2 and influenza viruses using influenza virus-like particles (VLP) incorporated by protein transfer with glycosylphosphatidylinositol (GPI)-anchored SARS-CoV-2 RBD fused to GM-CSF as an adjuvant. GPI-RBD-GM-CSF fusion protein was expressed in CHO-S cells, purified and incorporated onto influenza VLPs to develop the hybrid vaccine. Our results show that the hybrid vaccine induced a strong antibody response and protected mice from both influenza virus and mouse-adapted SARS-CoV-2 challenges, with vaccinated mice having significantly lower lung viral titers compared to naive mice. These results suggest that a hybrid vaccine strategy is a promising approach for developing multivalent vaccines to prevent influenza A and SARS-CoV-2 infections.

Background: PD-L1 is one of the major immune checkpoints which limits the effectiveness of antitumor immunity. Blockade of PD-L1/PD-1 has been a major improvement in the treatment of certain cancers, however, the response rate to checkpoint blockade remains low suggesting a need for new therapies. Metformin has emerged as a potential new drug for the treatment of cancer due to its effects on PD-L1 expression, T cell responses, and the immunosuppressive environment within tumors. While the benefits of metformin in combination with checkpoint blockade have been reported in animal models, little remains known about its effect on other types of immunotherapy., Methods: Vaccine immunotherapy and metformin were administered to mice inoculated with tumors to investigate the effect of metformin and TMV vaccine on tumor growth, metastasis, PD-L1 expression, immune cell infiltration, and CD8 T cell phenotype. The effect of metformin on IFN-γ induced PD-L1 expression in tumor cells was assessed by flow cytometry, western blot, and RT-qPCR., Results: We observed that tumors that respond to metformin and vaccine immunotherapy combination show a reduction in surface PD-L1 expression compared with tumor models that do not respond to metformin. In vitro assays showed that the effect of metformin on tumor cell PD-L1 expression was mediated in part by AMP-activated protein kinase signaling. Vaccination results in increased T cell infiltration in all tumor models, and this was not further enhanced by metformin. However, we observed an increased number of CD8 T cells expressing PD-1, Ki-67, Tim-3, and CD62L as well as increased effector cytokine production after treatment with metformin and tumor membrane vesicle vaccine., Conclusions: Our data suggest that metformin can synergize with vaccine immunotherapy to augment the antitumor response through tumor-intrinsic mechanisms and also alter the phenotype and function of CD8 T cells within the tumor, which could provide insights for its use in the clinic., Competing Interests: Competing interests: PS holds shares in Metaclipse Therapeutics Corporation, a company that is planning to use GPI-anchored molecules to develop membrane-based cancer vaccine in the future as suggested in the current manuscript., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Dendritic cells (DCs) are the most effective antigen presenting cells for the development of T cell responses. The only FDA approved DC-based immunotherapy to date is Sipuleucel-T, which utilizes a fusion protein to stimulate DCs ex vivo with GM-CSF and simultaneously deliver the antigen PAP for prostate cancer. This approach is restricted by the breadth of immunity elicited to a single antigen, and to cancers that have a defined tumor associated antigen. Other multi-antigen approaches have been restricted by poor efficacy of vaccine adjuvants. We have developed a vaccine platform that consists of autologous DCs pulsed with cytokine-adjuvanted tumor membrane vesicles (TMVs) made from tumor tissue, that encapsulate the antigenic landscape of individual tumors. Here we test the efficacy of DCs pulsed with TMVs incorporated with glycolipid-anchored immunostimulatory molecules (GPI-ISMs) in HER2-positive and triple negative breast cancer murine models. Pulsing of DCs with TMVs containing GPI-ISMs results in superior uptake of vesicles, DC activation and cytokine production. Adaptive transfer of TMV-pulsed DCs to tumor bearing mice results in the inhibition of tumor growth, reduction in lung metastasis, and an increase in immune cell infiltration into the tumors. These observations suggest that DCs pulsed with TMVs containing GPI-GM-CSF and GPI-IL-12 can be further developed to be used as a personalized immunotherapy platform for cancer treatment.

Triple-negative breast cancer (TNBC) afflicts women at a younger age than other breast cancers and is associated with a worse clinical outcome. This poor clinical outcome is attributed to a lack of defined targets and patient-to-patient heterogeneity in target antigens and immune responses. To address such heterogeneity, we tested the efficacy of a personalized vaccination approach for the treatment of TNBC using the 4T1 murine TNBC model. We isolated tumor membrane vesicles (TMVs) from homogenized 4T1 tumor tissue and incorporated glycosyl phosphatidylinositol (GPI)-anchored forms of the immunostimulatory B7-1 (CD80) and IL-12 molecules onto these TMVs to make a TMV vaccine. Tumor-bearing mice were then administered with the TMV vaccine either alone or in combination with immune checkpoint inhibitors. We show that TMV-based vaccine immunotherapy in combination with anti-CTLA-4 mAb treatment upregulated immunomodulatory cytokines in the plasma, significantly improved survival, and reduced pulmonary metastasis in mice compared to either therapy alone. The depletion of CD8 + T cells, but not CD4 + T cells, resulted in the loss of efficacy. This suggests that the vaccine acts via tumor-specific CD8 + T cell immunity. These results suggest TMV vaccine immunotherapy as a potential enhancer of immune checkpoint inhibitor therapies for metastatic triple-negative breast cancer.

Immune checkpoint inhibitor (ICI) immunotherapy improved the survival of head and neck squamous cell carcinoma (HNSCC) patients. However, more than 80% of the patients are still resistant to this therapy. To test whether the efficacy of ICI therapy can be improved by vaccine-induced immunity, we investigated the efficacy of a tumor membrane-based vaccine immunotherapy in murine models of HNSCC. The tumors, grown subcutaneously, are used to prepare tumor membrane vesicles (TMVs). TMVs are then incorporated with glycolipid-anchored immunostimulatory molecules GPI-B7-1 and GPI-IL-12 by protein transfer to generate the TMV vaccine. This TMV vaccine inhibited tumor growth and improved the survival of mice challenged with SCCVII tumor cells. The tumor-free mice survived for several months, remained tumor-free, and were protected following a secondary tumor cell challenge, suggesting that the TMV vaccine induced an anti-tumor immune memory response. However, no synergy with anti-PD1 mAb was observed in this model. In contrast, the TMV vaccine was effective in inhibiting MOC1 and MOC2 murine oral cancer models and synergized with anti-PD1 mAb in extending the survival of tumor-bearing mice. These observations suggest that tumor tissue based TMV vaccines can be harnessed to develop an effective personalized immunotherapy for HNSCC that can enhance the efficacy of immune checkpoint inhibitors.

Antigen delivered within particulate materials leads to enhanced antigen-specific immunity compared to soluble administration of antigen. However, current delivery approaches for antigen encapsulated in synthetic particulate materials are limited by the complexity of particle production that affects stability and immunogenicity of the antigen. Herein, we describe a protein delivery system that utilizes plasma membrane vesicles (PMVs) derived from biological materials such as cultured cells or isolated tissues and a simple protein transfer technology. We show that these particulate PMVs can be easily modified within 4 h by a protein transfer process to stably incorporate a glycosylphosphatidylinositol (GPI)-anchored form of the breast cancer antigen HER-2 onto the PMV surface. Immunization of mice with GPI-HER-2-modified-PMVs induced strong HER-2-specific antibody responses and protection from tumor challenge in two different breast cancer models. Further incorporation of the immunostimulatory molecules IL-12 and B7-1 onto the PMVs by protein transfer enhanced tumor protection and induced beneficial Th1 and Th2-type HER-2-specific immune responses. Since protein antigens can be easily converted to GPI-anchored forms, these results demonstrate that isolated plasma membrane vesicles can be modified with desired antigens along with immunostimulatory molecules by protein transfer and used as a vaccine delivery vehicle to elicit potent antigen-specific immunity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Repetitive Ag encounter, coupled with dynamic changes in Ag density and inflammation, imparts phenotypic and functional heterogeneity to memory virus-specific CD8 T cells in persistently infected hosts. For herpesvirus infections, which cycle between latency and reactivation, recent studies demonstrate that virus-specific T cell memory is predominantly derived from naive precursors recruited during acute infection. Whether functional memory T cells to viruses that persist in a nonlatent, low-level infectious state (smoldering infection) originate from acute infection-recruited naive T cells is not known. Using mouse polyomavirus (MPyV) infection, we previously showed that virus-specific CD8 T cells in persistently infected mice are stably maintained and functionally competent; however, a sizeable fraction of these memory T cells are short-lived. Further, we found that naive anti-MPyV CD8 T cells are primed de novo during persistent infection and contribute to maintenance of the virus-specific CD8 T cell population and its phenotypic heterogeneity. Using a new MPyV-specific TCR-transgenic system, we now demonstrate that virus-specific CD8 T cells recruited during persistent infection possess multicytokine effector function, have strong replication potential, express a phenotype profile indicative of authentic memory capability, and are stably maintained. In contrast, CD8 T cells recruited early in MPyV infection express phenotypic and functional attributes of clonal exhaustion, including attrition from the memory pool. These findings indicate that naive virus-specific CD8 T cells recruited during persistent infection contribute to preservation of functional memory against a smoldering viral infection.

Human polyomaviruses are associated with substantial morbidity in immunocompromised patients, including those with HIV/AIDS, recipients of bone marrow and kidney transplants, and individuals receiving immunomodulatory agents for autoimmune and inflammatory diseases. No effective antipolyomavirus agents are currently available, and no host determinants have been identified to predict susceptibility to polyomavirus-associated diseases. Using the mouse polyomavirus (MPyV) infection model, we recently demonstrated that perforin-granzyme exocytosis, tumor necrosis factor alpha (TNF-α), and Fas did not contribute to control of infection or virus-induced tumors. Gamma interferon (IFN-γ) was recently shown to inhibit replication by human BK polyomavirus in primary cultures of renal tubular epithelial cells. In this study, we provide evidence that IFN-γ is an important component of the host defense against MPyV infection and tumorigenesis. In immortalized and primary cells, IFN-γ reduces expression of MPyV proteins and impairs viral replication. Mice deficient for the IFN-γ receptor (IFN-γR(-/-)) maintain higher viral loads during MPyV infection and are susceptible to MPyV-induced tumors; this increased viral load is not associated with a defective MPyV-specific CD8(+) T cell response. Using an acute MPyV infection kidney transplant model, we further show that IFN-γR(-/-) donor kidneys harbor higher MPyV levels than donor kidneys from wild-type mice. Finally, administration of IFN-γ to persistently infected mice significantly reduces MPyV levels in multiple organs, including the kidney, a major reservoir for persistent mouse and human polyomavirus infections. These findings demonstrate that IFN-γ is an antiviral effector molecule for MPyV infection.

Virus-specific CD4(+) T cells optimize antiviral responses by providing help for antiviral humoral responses and CD8(+) T cell differentiation. Although CD4(+) T cell responses to viral infections that undergo complete clearance have been studied extensively, less is known about virus-specific CD4(+) T cell responses to viruses that persistently infect their hosts. Using a mouse polyomavirus (MPyV) infection model, we previously demonstrated that CD4(+) T cells are essential for recruiting naive MPyV-specific CD8(+) T cells in persistently infected mice. In this study, we defined two dominant MPyV-specific CD4(+) T cell populations, one directed toward an epitope derived from the nonstructural large T Ag and the other from the major viral capsid protein of MPyV. These MPyV-specific CD4(+) T cells vary in terms of their magnitude, functional profile, and phenotype during acute and persistent phases of infection. Using a minimally myeloablative-mixed bone marrow chimerism approach, we further show that naive virus-specific CD4(+) T cells, like anti-MPyV CD8(+) T cells, are primed de novo during persistent virus infection. In summary, these findings reveal quantitative and qualitative differences in the CD4(+) T cell response to a persistent virus infection and demonstrate that naive antiviral CD4(+) T cells are recruited during chronic polyomavirus infection.

MicroRNAs are small regulatory RNAs that post-transcriptionally regulate gene expression and can be encoded by viral as well as cellular genomes. The functions of most viral miRNAs are unknown and few have been studied in an in vivo context. Here we show that the murine polyomavirus (PyV) encodes a precursor microRNA that is processed into two mature microRNAs, both of which are active at directing the cleavage of the early PyV mRNAs. Furthermore, we identify a deletion mutant of polyomavirus that is defective in encoding the microRNAs. This mutant replicates normally and transforms cultured cells with efficiencies comparable to wildtype PyV. The miRNA mutant is competent to establish a transient infection of mice following parenteral inoculation, and is cleared post infection at approximately the same rate as the wildtype virus. In addition, under these laboratory conditions, we observe no differences in anti-viral CD8 T cell responses. These results indicate that PyV miRNA expression is not essential for infection of cultured cells or experimentally inoculated mice, and raise the possibility that its role in natural infection might involve aspects of acquisition or spread that are not recapitulated by experimental inoculation.

Herpes simplex virus-1 (HSV-1) represents a significant obstacle for vaccine designers, despite decades of investigation. The virus primarily infects the host at vulnerable mucosal surfaces that progresses to lesion development, latency in nervous tissue, and possible reactivation. Therefore, protection at the site of infection is crucial. Mucosal adjuvants are critical for the development of an effective vaccine approach and heat-shock protein 70 (Hsp70) represents an attractive candidate for this purpose. This study demonstrates that Hsp70 coupled to gB498-505 from HSV-1 induced mucosal and systemic priming of CD8(+) T cells capable of protecting C57BL/6 mice against a lethal vaginal challenge. Elevated gB-specific cytotoxicity was observed in the spleen of mice immunized with conjugated Hsp70 and gB498-505. In addition, both vaginal IFNgamma levels and viral clearance were enhanced in mice mucosally immunized with Hsp70 and gB peptide versus peptide only control mice or mice receiving Hsp70 and a control peptide. These studies demonstrate that Hsp70 can be used as an effective mucosal adjuvant capable of generating a protective cell-mediated immune response against HSV-1.

Although immunity against intracellular pathogens is primarily provided by CD8 T lymphocytes that recognize pathogen-derived peptides presented by major histocompatibility complex (MHC) class Ia molecules, MHC class Ib-restricted CD8 T cells have been implicated in antiviral immunity. Using mouse polyoma virus (PyV), we found that MHC class Ia-deficient (K(b-/-)D(b-/-)) mice efficiently control this persistently infecting mouse pathogen. CD8 T cell depletion mitigates clearance of PyV in K(b-/-)D(b-/-) mice. We identified the ligand for PyV-specific CD8 T cells in K(b-/-)D(b-/-) mice as a nonamer peptide from the VP2 capsid protein presented by Q9, a member of the beta(2) microglobulin-associated Qa-2 family. Using Q9-VP2 tetramers, we monitored delayed but progressive expansion of these antigen-specific CD8alphabeta T cells in K(b-/-)D(b-/-) mice. Importantly, we demonstrate that Q9-VP2-specific CD8 T cells more effectively clear wild-type PyV than a VP2 epitope(null) mutant PyV. Finally, we show that wild-type mice also generate Q9-restricted VP2 epitope-specific CD8 T cells to PyV infection. To our knowledge, this is the first evidence for a defined MHC class Ib-restricted antiviral CD8 T cell response that contributes to host defense. This study motivates efforts to uncover MHC class Ib-restricted CD8 T cell responses in other viral infections, and given the limited polymorphism of MHC class Ib molecules, it raises the possibility of developing peptide-based viral vaccines having broad coverage across MHC haplotypes.

The CD8 coreceptor is important for positive selection of major histocompatibility complex I (MHC-I)-restricted thymocytes and in the generation of pathogen-specific T cells. However, the requirement for CD8 in these processes may not be essential. We previously showed that mice lacking beta(2)-microglobulin are highly susceptible to tumors induced by mouse polyoma virus (PyV), but CD8-deficient mice are resistant to these tumors. In this study, we show that CD8-deficient mice also control persistent PyV infection as efficiently as wild-type mice and generate a substantial virus-specific, MHC-I-restricted, T-cell response. Infection with vesicular stomatitis virus (VSV), which is acutely cleared, also recruited antigen-specific, MHC-I-restricted T cells in CD8-deficient mice. Yet, unlike in VSV infection, the antiviral MHC-I-restricted T-cell response to PyV has a prolonged expansion phase, indicating a requirement for persistent infection in driving T-cell inflation in CD8-deficient mice. Finally, we show that the PyV-specific, MHC-I-restricted T cells in CD8-deficient mice, while maintained long term at near-wild-type levels, are short lived in vivo and have extremely narrow T-cell receptor repertoires. These findings provide a possible explanation for the resistance of CD8-deficient mice to PyV-induced tumors and have implications for the maintenance of virus-specific MHC-I-restricted T cells during persistent infection.

Although many studies have investigated the requirement for CD4(+) T cell help for CD8(+) T cell responses to acute viral infections that are fully resolved, less is known about the role of CD4(+) T cells in maintaining ongoing CD8(+) T cell responses to persistently infecting viruses. Using mouse polyoma virus (PyV), we asked whether CD4(+) T cell help is required to maintain antiviral CD8(+) T cell and humoral responses during acute and persistent phases of infection. Though fully intact during acute infection, the PyV-specific CD8(+) T cell response declined numerically during persistent infection in MHC class II-deficient mice, leaving a small antiviral CD8(+) T cell population that was maintained long term. These unhelped PyV-specific CD8(+) T cells were functionally unimpaired; they retained the potential for robust expansion and cytokine production in response to Ag rechallenge. In addition, although a strong antiviral IgG response was initially elicited by MHC class II-deficient mice, these Ab titers fell, and long-lived PyV-specific Ab-secreting cells were not detected in the bone marrow. Finally, using a minimally myeloablative mixed bone marrow chimerism approach, we demonstrate that recruitment and/or maintenance of new virus-specific CD8(+) T cells during persistent infection is impaired in the absence of MHC class II-restricted T cells. In summary, these studies show that CD4(+) T cells differentially affect CD8(+) T cell responses over the course of a persistent virus infection.

Chronic Ag exposure during persistent viral infection erodes virus-specific CD8 T cell numbers and effector function, with a concomitant loss of pathogen control. Less clear are the respective contributions of Ag-specific and Ag-nonspecific (bystander) events on the quantity, quality, and maintenance of antiviral CD8 T cells responding to persistent virus infection. In this study, we show that low-dose inoculation with mouse polyomavirus (PyV) elicits a delayed, but numerically equivalent, antiviral CD8 T cell response compared with high-dose inoculation. Low-dose infection generated virus-specific CD8 T cells endowed with multicytokine functionality and a superior per cell capacity to produce IFN-gamma. PyV-specific CD8 T cells primed by low-dose inoculation also expressed higher levels of IL-7Ralpha and bcl-2 and possessed enhanced Ag-independent survival. Importantly, the quantity and quality of the antiviral CD8 T cell response elicited by dendritic cell-mediated immunization were mitigated by infection with a mutant PyV lacking the dominant CD8 T cell viral epitope. These findings suggest that the fitness of the CD8 T cell response to persistent virus infection is programmed in large part by early virus-associated Ag-nonspecific factors, and imply that limiting bystander inflammation at the time of inoculation, independent of Ag load, may optimize adaptive immunity to persistent viral infection.

[structure: see text] A family of cyclic 1-deoxysphingolipid derivatives of structure 4 has been designed and synthesized, which may serve as tumorigenesis suppressors for various cancers. Compound 4 is a second-generation analogue developed from sphingosine (1), in which a hydroxyl substituent is moved from C1 to C5 and a methylene is added for conformational rigidity between the C2-nitrogen substituent and C4. The synthetic chemistry for pyrrolidine ring closure at C3-C4 features ring-closing metathesis followed by hydroboration-oxidation.

Immunization of the neonate is a highly desirable goal for vaccine developers, since the neonate is profoundly susceptible to a number of viral and bacterial pathogens. The neonatal immune system tends to generate Th2 recall responses, known as neonatal tolerance, which may not protect against viral challenge later in life. In this study we demonstrate that a potent immune proinflammatory stimulator, heat-shock protein 70 (hsp70), can act as an effective and safe adjuvant in neonates. Priming of neonates with hsp70 coupled to a viral MHC Class I-restricted epitope (gB498-505) and injection with recombinant gB generated strong cytotoxic T lymphocyte (CTL) responses and a Th1 primary T helper cell response during the neonatal period. In addition, enhanced CTL and predominant Th1 recall responses to viral antigens were observed following secondary challenge as adults. These responses were sufficient to allow protection against a lethal challenge with Herpes Simplex Virus Type-1 (HSV-1). Therefore, hsp70 in conjunction with viral epitopes and recombinant viral protein can perhaps prime protective immune responses to herpes viruses early in life when infection, which can be life-threatening, and the establishment of latency frequently occur.

Mucosal surfaces represent the entry route of a multitude of viral pathogens. For many of these viruses, such as the herpes simplex viruses and human immunodeficiency virus, no effective vaccine exists. Hence, it is important that prospective vaccines engender maximal immunity at these susceptible sites. Genetic vaccines encoding adjuvant molecules represent one approach to optimize mucosal as well as systemic immunity. Promising candidates include various inflammatory cytokines and chemokines that might be used to enhance the primary response to a level sufficient for protection. Encouraging studies involving cytokines such as granulocyte/macrophage colony-stimulating factor, interleukin-2 (IL-2), IL-12, IL-18, and many others are examined. Notable chemokines that may offer hope in such efforts include IL-8, RANTES, CCL19, CCL21, and a few others. Combinatorial approaches utilizing several cytokines and chemokines will most likely yield the greatest success. In addition, as more is discovered regarding the requirements for memory development of T cells, boosters involving key cytokines such as IL-15 and IL-23 may prove beneficial to long-term maintenance of the memory pool. This review summarizes the progress in the use of genetic vaccines to achieve mucosal immunity and discusses the needed strategies to maximize long-term prospective immunity at this vulnerable entry site.

Naturally occurring CD4+CD25+ regulatory T cells appear important to prevent activation of autoreactive T cells. This article demonstrates that the magnitude of a CD8+ T cell-mediated immune response to an acute viral infection is also subject to control by CD4+CD25+ T regulatory cells (Treg). Accordingly, if natural Treg were depleted with specific anti-CD25 antibody before infection with HSV, the resultant CD8+ T cell response to the immunodominant peptide SSIEFARL was significantly enhanced. This was shown by several in vitro measures of CD8+ T cell reactivity and by assays that directly determine CD8+ T cell function, such as proliferation and cytotoxicity in vivo. The enhanced responsiveness in CD25-depleted animals was between three- and fourfold with the effect evident both in the acute and memory phases of the immune response. Surprisingly, HSV infection resulted in enhanced Treg function with such cells able to suppress CD8+ T cell responses to both viral and unrelated antigens. Our results are discussed both in term of how viral infection might temporarily diminish immunity to other infectious agents and their application to vaccines. Thus, controlling suppressor effects at the time of vaccination could result in more effective immunity.

The report shows that CpG can exert additional adjuvant effects by inducing cells that are normally inferior antigen (Ag)-presenting cells to participate in immune induction by cross-priming. Macrophages (Mphi) exposed to protein Ag in the presence of bioactive CpG DNA released material that induced primary CD8(+) T cell responses in DC-naïve T cell cultures. This cross-priming event was accompanied by up-regulation of the stress protein response as well as inflammatory cytokine expression in treated Mphi. The material released was indicated to contain inducible heat shock protein-70 and epitope peptide, which in turn, were presented by dendritic cells (DCs) to responder T cells. Such an adjuvant effect by CpG may serve to salvage immunogenic material from otherwise inert depot cellular sites and additionally stimulate DCs to effectively cross-prime. The cross-priming, shown also to occur in vivo, may be particularly useful when Ag doses are low and have minimal opportunity for delivery to DCs for consequent direct priming.

Neonatal exposure to Ag has always been considered suppressive for immunity. Recent investigations, however, indicated that the neonatal immune system could be guided to develop immunity. For instance, delivery of a proteolipid protein (PLP) peptide on Ig boosts the neonatal immune system to develop responses upon challenge with the PLP peptide later. Accordingly, mice given Ig-PLP at birth and challenged with the PLP peptide as adults developed proliferative T cells in the lymph node that produced IL-4 instead of the usual Th1 cytokines. However, the spleen was unresponsive unless IL-12 was provided. Herein, we wished to determine whether such a neonatal response is intrinsic to the PLP peptide or could develop with an unrelated myelin peptide as well as whether the T cell deviation is able to confer resistance to autoimmunity involving diverse T cell specificities. Accordingly, the amino acid sequence 87-99 of myelin basic protein was expressed on the same Ig backbone, and the resulting Ig-myelin basic protein chimera was tested for induction of neonatal immunity and protection against experimental allergic encephalomyelitis. Surprisingly, the results indicated that immunity developed in the lymph node and spleen, with deviation of T cells occurring in both organs. More striking, the splenic T cells produced IL-10 in addition to IL-4, providing an environment that facilitated bystander deviation of responses to unrelated epitopes and promoted protection against experimental allergic encephalomyelitis involving diverse T cell specificities. Thus, neonatal exposure to Ag can prime responses in various organs and sustain regulatory functions effective against diverse autoreactive T cells.

Autoimmunity arises when the immune system no longer tolerates self and precipitates lymphocyte reactivity against our own antigens. Although the developing T cell repertoire is constantly purging, self-recognition events do exist when such tight control is evaded and autoreactive lymphocytes escape the thymus (the sites of T cell development) and migrate to the periphery. Upon activation these autoreactive cells may exert aggressive behavior toward one's own tissues and organs leading to autoimmune disease. Multiple sclerosis, Rheumatoid arthritis, and type I diabetes are autoimmune diseases mediated by autoreactive T cells. A logical approach to prevent such autoimmunity would be to reprogram those lymphocytes to tolerate the self antigen. Injection of antigen at the neonatal stage promotes a state of tolerance such that successive encounter with antigen does not precipitate aggressive reactions. The mechanism underlying neonatal tolerance involves priming of T cells whose effector functions do not cause inflammatory reactions upon recognition of antigen but rather induce protective immunity. This form of tolerant immunity provides an attractive strategy for vaccination against autoimmunity. Herein, it is shown that neonatal exposure to a self-peptide-immunoglobulin chimera drives a tolerant immunity toward the self-peptide and protects against the autoimmune disease, experimental allergic encephalomyelitis.

Ig-PLP1 and Ig-PLP-LR are chimeric Igs expressing proteolipid protein (PLP)-derived T cell agonist (PLP1) and antagonist (PLP-LR) peptides, respectively. Both chimeras, like free PLP1 and PLP-LR peptides, induce in vivo-specific T cell responses. However, the responses induced by Ig-PLP1 and Ig-PLP-LR were cross-reactive with both PLP1 and PLP-LR peptides, while those induced by free peptides were not. Surprisingly, despite the cross-reactivity of the responses, when Ig-PLP1 and Ig-PLP-LR were administered together into mice, a dose-dependent down-regulation of both T cell responses and a reduction of IL-2 production to background levels was observed. In contrast, when T cells induced by either Ig chimera were stimulated in vitro with mixtures of free PLP1 and PLP-LR peptides, there was no down-regulation of proliferation or decrease in IL-2 production. These data indicate that Ig-PLP1 and Ig-PLP-LR exert adverse reactions on one another at the level of naive T cells, resulting in an opposite antagonism. However, naive T cells experiencing either chimera develop into cross-reactive cells, acquire resistance to TCR triggering by closely related but different peptides, and support responsiveness.