MHC class II-associated invariant chain linkage of antigen dramatically improves cell-mediated immunity induced by adenovirus vaccines

The ideal vaccine induces a potent protective immune response, which should be rapidly induced, long-standing, and of broad specificity. Recombinant adenoviral vectors induce potent Ab and CD8+ T cell responses against transgenic Ags within weeks of administration, and they are among the most potent and versatile Ag delivery vehicles available. However, the impact of chronic infections like HIV and hepatitis C virus underscore the need for further improvements. In this study, we show that the protective immune response to an adenovirus-encoded vaccine Ag can be accelerated, enhanced, broadened, and prolonged by tethering of the rAg to the MHC class II-associated invariant chain (Ii). Thus, adenovirus-vectored vaccines expressing lymphocytic choriomeningitis virus (LCMV)-derived glycoprotein linked to Ii increased the CD4+ and CD8+ T cell stimulatory capacity in vitro and in vivo. Furthermore, mice vaccinated with a single dose of adenovirus-expressing LCMV-derived glycoprotein linked to Ii were protected against lethal virus-induced choriomeningitis, lethal challenge with strains mutated in immunodominant T cell epitopes, and systemic infection with a highly invasive strain. In therapeutic tumor vaccination, the vaccine was as efficient as live LCMV. In comparison, animals vaccinated with a conventional adenovirus vaccine expressing unmodified glycoprotein were protected against systemic infection, but only temporarily against lethal choriomeningitis, and this vaccine was less efficient in tumor therapy.     

Induction of humoral and CD8+ T cell responses are required for protection against lethal Ebola virus infection

Ebola virus (EBOV)-like particles (eVLP), composed of the EBOV glycoprotein and matrix viral protein (VP)40 with a lipid membrane, are a highly efficacious method of immunization against EBOV infection. The exact requirements for immunity against EBOV infection are poorly defined at this time. The goal of this work was to determine the requirements for EBOV immunity following eVLP vaccination. Vaccination of BALB/c or C57BL/6 mice with eVLPs in conjunction with QS-21 adjuvant resulted in mixed IgG subclass responses, a Th1-like memory cytokine response, and protection from lethal EBOV challenge. Further, this vaccination schedule led to the generation of both CD4(+) and CD8(+) IFN-gamma(+) T cells recognizing specific peptides within glycoprotein and VP40. The transfer of both serum and splenocytes, but not serum or splenocytes alone, from eVLP-vaccinated mice conferred protection against lethal EBOV infection in these studies. B cells were required for eVLP-mediated immunity to EBOV because B cell-deficient mice vaccinated with eVLPs were not protected from lethal EBOV challenge. We also found that CD8(+), but not CD4(+), T cells are absolutely required for eVLP-mediated protection against EBOV infection. Further, eVLP-induced protective mechanisms were perforin-independent, but IFN-gamma-dependent. Taken together, both EBOV-specific humoral and cytotoxic CD8(+) T cell responses are critical to mediate protection against filoviruses following eVLP vaccination.     

An MHC class Ib-restricted CD8+ T cell response to lymphocytic choriomeningitis virus

Conventional MHC class Ia-restricted CD8(+) T cells play a dominant role in the host response to virus infections, but recent studies indicate that T cells with specificity for nonclassical MHC class Ib molecules may also participate in host defense. To investigate the potential role of class Ib molecules in anti-viral immune responses, K(b-/-)D(b-/-)CIITA(-/-) mice lacking expression of MHC class Ia and class II molecules were infected with lymphocytic choriomeningitis virus (LCMV). These animals have a large class Ib-selected CD8(+) T cell population and they were observed to mediate partial (but incomplete) virus clearance during acute LCMV infection as compared with K(b-/-)D(b-/-)β(2)-microglobulin(-/-) mice that lack expression of both MHC class Ia and class Ib molecules. Infection was associated with expansion of splenic CD8(+) T cells and induction of granzyme B and IFN-γ effector molecules in CD8(+) T cells. Partial virus clearance was dependent on CD8(+) cells. In vitro T cell restimulation assays demonstrated induction of a population of β(2)-microglobulin-dependent, MHC class Ib-restricted CD8(+) T cells with specificity for viral Ags and yet to be defined nonclassical MHC molecules. MHC class Ib-restricted CD8(+) T cell responses were also observed after infection of K(b-/-)D(b-/-)mice despite the low number of CD8(+) T cells in these animals. Long-term infection studies demonstrated chronic infection and gradual depletion of CD8(+) T cells in K(b-/-)D(b-/-)CIITA(-/-) mice, demonstrating that class Ia molecules are required for viral clearance. These findings demonstrate that class Ib-restricted CD8(+) T cells have the potential to participate in the host immune response to LCMV.     

CD8+ cellular immunity mediates rAd5 vaccine protection against Ebola virus infection of nonhuman primates

Vaccine-induced immunity to Ebola virus infection in nonhuman primates (NHPs) is marked by potent antigen-specific cellular and humoral immune responses; however, the immune mechanism of protection remains unknown. Here we define the immune basis of protection conferred by a highly protective recombinant adenovirus virus serotype 5 (rAd5) encoding Ebola virus glycoprotein (GP) in NHPs. Passive transfer of high-titer polyclonal antibodies from vaccinated Ebola virus-immune cynomolgus macaques to naive macaques failed to confer protection against disease, suggesting a limited role of humoral immunity. In contrast, depletion of CD3(+) T cells in vivo after vaccination and immediately before challenge eliminated immunity in two vaccinated macaques, indicating a crucial requirement for T cells in this setting. The protective effect was mediated largely by CD8(+) cells, as depletion of CD8(+) cells in vivo using the cM-T807 monoclonal antibody (mAb), which does not affect CD4(+) T cell or humoral immune responses, abrogated protection in four out of five subjects. These findings indicate that CD8(+) cells have a major role in rAd5-GP-induced immune protection against Ebola virus infection in NHPs. Understanding the immunologic mechanism of Ebola virus protection will facilitate the development of vaccines for Ebola and related hemorrhagic fever viruses in humans.     

Protective cytotoxic T-cell responses induced by venezuelan equine encephalitis virus replicons expressing Ebola virus proteins

Infection with Ebola virus causes a severe disease accompanied by high mortality rates, and there are no licensed vaccines or therapies available for human use. Filovirus vaccine research efforts still need to determine the roles of humoral and cell-mediated immune responses in protection from Ebola virus infection. Previous studies indicated that exposure to Ebola virus proteins expressed from packaged Venezuelan equine encephalitis virus replicons elicited protective immunity in mice and that antibody-mediated protection could only be demonstrated after vaccination against the glycoprotein. In this study, the murine CD8(+) T-cell responses to six Ebola virus proteins were examined. CD8(+) T cells specific for Ebola virus glycoprotein, nucleoprotein, and viral proteins (VP24, VP30, VP35, and VP40) were identified by intracellular cytokine assays using splenocytes from vaccinated mice. The cells were expanded by restimulation with peptides and demonstrated cytolytic activity. Adoptive transfer of the CD8(+) cytotoxic T cells protected filovirus na?ve mice from challenge with Ebola virus. These data support a role for CD8(+) cytotoxic T cells as part of a protective mechanism induced by vaccination against six Ebola virus proteins and provide additional evidence that cytotoxic T-cell responses can contribute to protection from filovirus infections.     

Identification of novel HLA-DR1-restricted epitopes from the hepatitis B virus envelope protein in mice expressing HLA-DR1 and vaccinated human subjects

Helper T lymphocytes that control CD8(+) T-cell and antibody responses are key elements for the resolution of infection by the hepatitis B virus and for the development of effective immunological memory after hepatitis B vaccination. We have used H-2 class II-deficient mice that express the human MHC class II molecule, HLA-DR1, to identify novel hepatitis B virus envelope-derived T helper epitopes. We confirmed the immunogenicity of a previously described HLA-DR1-restricted epitope, and identified three novel epitopes. CD4(+) T-cell immune responses against these epitopes were detected in peripheral blood mononuclear cells from HLA-DR1(+) individuals vaccinated against hepatitis B. We showed that subjects receiving the currently available hepatitis B vaccines do not develop cross-reactive T helper responses against one of the novel epitopes which are structurally variable between different hepatitis B virus subtypes. These findings highlight the need for developing vaccines against a wider range of viral subtypes, and establish humanized mice as a convenient tool for identifying new immunogenic epitopes from pathogens.     

Critical contribution of immunoproteasomes in the induction of protective immunity against Trypanosoma cruzi in mice vaccinated with a plasmid encoding a CTL epitope fused to green fluorescence protein

Acquired immunity against infection with Trypanosoma cruzi is dependent on CD8(+)T cells. Here, to develop a vaccine strategy taking advantage of activated CD8(+)T cells, we constructed a DNA vaccine, designated pGFP-TSA1, encoding a fusion protein linking GFP to a single CTL epitope of TSA1, a leading candidate for vaccine against T. cruzi. C57BL/6 mice vaccinated with this plasmid showed suppressed parasitemia and prolonged survival. Vaccination with pGFP-TSA1 enhanced epitope-specific cytotoxicity and IFN-gamma secretion by CD8(+)T cells. Furthermore, the depletion of CD8(+)T cells prior to challenge infection with T. cruzi completely abolished this protection, indicating that CD8(+)T cells are the principal effector T cells involved. When mice deficient in the proteasome activator PA28alpha/beta or the immunoproteasome subunits LMP2 and LMP7 were used, the protective immunity against infection was profoundly attenuated. Our findings clearly demonstrate that vaccination with pGFP-TSA1 successfully induces protection dependent on CD8(+)T cell activation, in which immunoproteasomes play a crucial role. It is noteworthy to document that physical binding of the epitope and GFP is required for induction of this protection, since mice vaccinated with pTSA1-IRES-GFP failed to acquire resistance, probably because the epitope and GFP are separately expressed in the antigen-presenting cells.     

Induction of in vivo functional Db-restricted cytolytic T cell activity against a putative phosphate transport receptor of Mycobacterium tuberculosis

Using plasmid vaccination with DNA encoding the putative phosphate transport receptor PstS-3 from Mycobacterium tuberculosis and 36 overlapping 20-mer peptides spanning the entire PstS-3 sequence, we determined the immunodominant Th1-type CD4(+) T cell epitopes in C57BL/10 mice, as measured by spleen cell IL-2 and IFN-gamma production. Furthermore, a potent IFN-gamma-inducing, D(b)-restricted CD8(+) epitope was identified using MHC class I mutant B6.C-H-2(bm13) mice and intracellular IFN-gamma and whole blood CD8(+) T cell tetramer staining. Using adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA vaccinated or M. tuberculosis-infected recipients, we demonstrated a functional in vivo CTL activity against this D(b)-restricted PstS-3 epitope. IFN-gamma ELISPOT responses to this epitope were also detected in tuberculosis-infected mice. The CD4(+) and CD8(+) T cell epitopes defined for PstS-3 were completely specific and not recognized in mice vaccinated with either PstS-1 or PstS-2 DNA. The H-2 haplotype exerted a strong influence on immune reactivity to the PstS-3 Ag, and mice of the H-2(b, p, and f) haplotype produced significant Ab and Th1-type cytokine levels, whereas mice of H-2(d, k, r, s, and q) haplotype were completely unreactive. Low responsiveness against PstS-3 in MHC class II mutant B6.C-H-2(bm12) mice could be overcome by DNA vaccination. IFN-gamma-producing CD8(+) T cells could also be detected against the D(b)-restricted epitope in H-2(p) haplotype mice. These results highlight the potential of DNA vaccination for the induction and characterization of CD4(+) and particularly CD8(+) T cell responses against mycobacterial Ags.     

Single-epitope DNA vaccination prevents exhaustion and facilitates a broad antiviral CD8+ T cell response during chronic viral infection

Induction of a monospecific antiviral CD8+ T cell response may pose a risk to the host due to the narrow T cell response induced. At the individual level, this may result in selection of CD8+ T cell escape variants, particularly during chronic viral infection. Second, prior immunization toward a single dominant epitope may suppress the response to other viral epitopes, and this may lead to increased susceptibility to reinfection with escape variants circulating in the host population. To address these issues, we induced a memory response consisting solely of monospecific, CD8+ T cells by use of DNA vaccines encoding immunodominant epitopes of lymphocytic choriomeningitis virus (LCMV). We analyzed the spectrum of the CD8+ T cell response and the susceptibility to infection in H-2(b) and H-2(d) mice. Priming for a monospecific, CD8+ T cell response did not render mice susceptible to viral variants. Thus, vaccinated mice were protected against chronic infection with LCMV, and no evidence indicating biologically relevant viral escape was obtained. In parallel, a broad and sustained CD8+ T cell response was generated upon infection, and in H-2(d) mice epitope spreading was observed. Even after acute LCMV infection, DNA vaccination did not significantly impair naturally induced immunity. Thus, the response to the other immunogenic epitopes was not dramatically suppressed in DNA-immunized mice undergoing normal immunizing infection, and the majority of mice were protected against rechallenge with escape variants. These findings underscore that a monospecific vaccine may induce efficient protective immunity given the right set of circumstances.     

Direct ex vivo kinetic and phenotypic analyses of CD8(+) T-cell responses induced by DNA immunization

CD8(+) T-cell responses can be induced by DNA immunization, but little is known about the kinetics of these responses in vivo in the absence of restimulation or how soon protective immunity is conferred by a DNA vaccine. It is also unclear if CD8(+) T cells primed by DNA vaccines express the vigorous effector functions characteristic of cells primed by natural infection or by immunization with a recombinant live virus vaccine. To address these issues, we have used the sensitive technique of intracellular cytokine staining to carry out direct ex vivo kinetic and phenotypic analyses of antigen-specific CD8(+) T cells present in the spleens of mice at various times after (i) a single intramuscular administration of a plasmid expressing the nucleoprotein (NP) gene from lymphocytic choriomeningitis virus (LCMV), (ii) infection by a recombinant vaccinia virus carrying the same protein (vvNP), or (iii) LCMV infection. In addition, we have evaluated the rapidity with which protective immunity against both lethal and sublethal LCMV infections is achieved following DNA vaccination. The CD8(+) T-cell response in DNA-vaccinated mice was slightly delayed compared to LCMV or vvNP vaccinees, peaking at 15 days postimmunization. Interestingly, the percentage of antigen-specific CD8(+) T cells present in the spleen at day 15 and later time points was similar to that observed following vvNP infection. T cells primed by DNA vaccination or by infection exhibited similar cytokine expression profiles and had similar avidities for an immunodominant cytotoxic T lymphocyte epitope peptide, implying that the responses induced by DNA vaccination differ quantitatively but not qualitatively from those induced by live virus infection. Surprisingly, protection from both lethal and sublethal LCMV infections was conferred within 1 week of DNA vaccination, well before the peak of the CD8(+) T-cell response.     

Efficient lung recruitment of respiratory syncytial virus-specific Th1 cells induced by recombinant bacillus Calmette-Guérin promotes virus clearance and protects from infection

Infection by the respiratory syncytial virus (RSV) can cause extensive inflammation and lung damage in susceptible hosts due to a Th2-biased immune response. Such a deleterious inflammatory response can be enhanced by immunization with formalin- or UV-inactivated RSV, as well as with vaccinia virus expressing the RSV-G protein. Recently, we have shown that vaccination with rBCG-expressing RSV Ags can prevent the disease in the mouse. To further understand the immunological mechanisms responsible for protection against RSV, we have characterized the T cell populations contributing to virus clearance in mice immunized with this BCG-based vaccine. We found that both CD4(+) and CD8(+) T cells were recruited significantly earlier to the lungs of infected mice that were previously vaccinated. Furthermore, we observed that simultaneous adoptive transfer of CD8(+) and CD4(+) RSV-specific T cells from vaccinated mice was required to confer protection against virus infection in naive recipients. In addition, CD4(+) T cells induced by vaccination released IFN-γ after RSV challenge, indicating that protection is mediated by a Th1 immune response. These data suggest that vaccination with rBCG-expressing RSV Ags can induce a specific effector/memory Th1 immune response consisting on CD4(+) and CD8(+) T cells, both necessary for a fully protective response against RSV. These results support the notion that an effective induction of Th1 T cell immunity against RSV during childhood could counteract the unbalanced Th2-like immune response triggered by the natural RSV infection.     

The antiviral CD8+ T cell response is differentially dependent on CD4+ T cell help over the course of 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.     

A CD8+ T cell heptaepitope minigene vaccine induces protective immunity against Chlamydia pneumoniae

An intact T cell compartment and IFN-gamma signaling are required for protective immunity against Chlamydia. In the mouse model of Chlamydia pneumoniae (Cpn) infection, this immunity is critically dependent on CD8(+) T cells. Recently we reported that Cpn-infected mice generate an MHC class I-restricted CD8(+) Tc1 response against various Cpn Ags, and that CD8(+) CTL to multiple epitopes inhibit Cpn growth in vitro. Here, we engineered a DNA minigene encoding seven H-2(b)-restricted Cpn CTL epitopes, the universal pan-DR epitope Th epitope, and an endoplasmic reticulum-translocating signal sequence. Immunization of C57BL/6 mice with this construct primed IFN-gamma-producing CD8(+) CTL against all seven CTL epitopes. CD8(+) T cell lines generated to minigene-encoded CTL epitopes secreted IFN-gamma and TNF-alpha and exhibited CTL activity upon recognition of Cpn-infected macrophages. Following intranasal challenge with Cpn, a 3.6 log reduction in mean lung bacterial numbers compared with control animals was obtained. Using a 20-fold increase in the Cpn challenging dose, minigene-vaccinated mice had a 60-fold reduction in lung bacterial loads, compared with controls. Immunization and challenge studies with beta(2)-microglobulin(-/-) mice indicated that the reduction of lung Cpn burdens was mediated by the MHC class I-dependent CD8(+) T cells to minigene-included Cpn CTL epitopes, rather than by pan-DR epitope-specific CD4(+) T cells. This constitutes the first demonstration of significant protection achieved by immunization with a CD8(+) T cell epitope-based DNA construct in a bacterial system and provides the basis for the optimal design of multicomponent anti-Cpn vaccines for humans.     

Peripheral CD8+CD25+ T lymphocytes from MHC class II-deficient mice exhibit regulatory activity

We characterized CD8(+) T cells constitutively expressing CD25 in mice lacking the expression of MHC class II molecules. We showed that these cells are present not only in the periphery but also in the thymus. Like CD4(+)CD25(+) T cells, CD8(+)CD25(+) T cells appear late in the periphery during ontogeny. Peripheral CD8(+)CD25(+) T cells from MHC class II-deficient mice also share phenotypic and functional features with regulatory CD4(+)CD25(+) T cells: in particular, they strongly express glucocorticoid-induced TNFR family-related gene, CTLA-4 and Foxp3, produce IL-10, and inhibit CD25(-) T cell responses to anti-CD3 stimulation through cell contacts with similar efficiency to CD4(+)CD25(+) T cells. However, unlike CD4(+)CD25(+) T cells CD8(+)CD25(+) T cells from MHC class II-deficient mice strongly proliferate and produce IFN-gamma in vitro in response to stimulation in the absence of exogenous IL-2.     

DNA vaccination against persistent viral infection.

This study shows that DNA vaccination can confer protection against a persistent viral infection by priming CD8+ cytotoxic T lymphocytes (CTL). Adult BALB/c (H-2d) mice were injected intramuscularly with a plasmid expressing the nucleoprotein (NP) gene of lymphocytic choriomeningitis virus (LCMV) under the control of the cytomegalovirus promoter. The LCMV NP contains the immunodominant CTL epitope (amino acids 118 to 126) recognized by mice of the H-2d haplotype. After three injections with 200 micrograms of NP DNA, the vaccinated mice were challenged with LCMV variants (clones 13 and 28b) that establish persistent infection in naive adult mice. Fifty percent of the DNA-vaccinated mice were protected, as evidenced by decreased levels of infectious virus in the blood and tissues, eventual clearance of viral antigen from all organs tested, the presence of an enhanced LCMV-specific CD8+ CTL response, and maintenance of memory CTL after clearance of virus infection. However, it should be noted that protection was seen in only half of the vaccinated mice, and we were unable to directly measure virus-specific immune responses in any of the DNA-vaccinated mice prior to LCMV challenge. Thus, at least in the system that we have used, gene immunization was a suboptimal method of inducing protective immunity and was several orders of magnitude less efficient than vaccination with live virus. In conclusion, our results show that DNA immunization works against a persistent viral infection but that efforts should be directed towards improving this novel method of vaccination.     

Dendritic cells charged with apoptotic tumor cells induce long-lived protective CD4+ and CD8+ T cell immunity against B16 melanoma

Dendritic cells (DCs) are potent APCs and attractive vectors for cancer immunotherapy. Using the B16 melanoma, a poorly immunogenic experimental tumor that expresses low levels of MHC class I products, we investigated whether DCs loaded ex vivo with apoptotic tumor cells could elicit combined CD4(+) and CD8(+) T cell dependent, long term immunity following injection into mice. The bone marrow-derived DCs underwent maturation during overnight coculture with apoptotic melanoma cells. Following injection, DCs migrated to the draining lymph nodes comparably to control DCs at a level corresponding to approximately 0.5% of the injected inoculum. Mice vaccinated with tumor-loaded DCs were protected against an intracutaneous challenge with B16, with 80% of the mice remaining tumor-free 12 wk after challenge. CD4(+) and CD8(+) T cells were efficiently primed in vaccinated animals, as evidenced by IFN-gamma secretion after in vitro stimulation with DCs loaded with apoptotic B16 or DCs pulsed with the naturally expressed melanoma Ag, tyrosinase-related protein 2. In addition, B16 melanoma cells were recognized by immune CD8(+) T cells in vitro, and cytolytic activity against tyrosinase-related protein 2(180-188)-pulsed target cells was observed in vivo. When either CD4(+) or CD8(+) T cells were depleted at the time of challenge, the protection was completely abrogated. Mice receiving a tumor challenge 10 wk after vaccination were also protected, consistent with the induction of tumor-specific memory. Therefore, DCs loaded with cells undergoing apoptotic death can prime melanoma-specific helper and CTLs and provide long term protection against a poorly immunogenic tumor in mice.     

Cellular and humoral immunity against vaccinia virus infection of mice

Despite the widespread use of vaccinia virus (VV) as a vector for other Ags and as the smallpox vaccine, there is little information available about the protective components of the immune response following VV infection. In this study, protection against wild-type VV was evaluated in mice with respect to the relative contributions of CD8(+) T cells vs that of CD4(+) T cells and Ab. C57BL/6 mice primed with the Western Reserve strain of VV mount significant IgM and IgG Ab responses, specific cytotoxic T cell responses, IFN-gamma responses in CD4(+) and CD8(+) T cells, and effectively clear the virus. This protection was abrogated by in vivo depletion of CD4(+) T cells or B cells in IgH(-/-) mice, but was not sensitive to CD8(+) T cell depletion alone. However, a role for CD8(+) T cells in primary protection was demonstrated in MHC class II(-/-) mice, where depleting CD8(+) T cells lead to increase severity of disease. Unlike control MHC class II(-/-) mice, the group depleted of CD8(+) T cells developed skin lesions on the tail and feet and had adrenal necrosis. Adoptive transfer experiments also show CD8(+) T cells can mediate protective memory. These results collectively show that both CD4(+) and CD8(+) T cell-mediated immunity can contribute to protection against VV infection. However, CD4(+) T cell-dependent anti-virus Ab production plays a more important role in clearing virus following acute infection, while in the absence of Ab, CD8(+) T cells can contribute to protection against disease.     

Rejection of intraocular tumors by CD4(+) T cells without induction of phthisis.

Immune privilege of the eye protects against sight-threatening inflammatory events, but can also permit outgrowth of otherwise nonlethal immunogenic tumors. Nonetheless, ocular tumor growth can be controlled by cellular immune responses. However, this will normally result in phthisis of the eye, in case tumor rejection is mediated by a delayed-type hypersensitivity response orchestrated by CD4(+) T cells. We now show that intraocular tumors can be eradicated by CD4(+) Th cells without inducing collateral damage of neighboring ocular tissue. Injection of tumor cells transformed by the early region 1 of human adenovirus type 5 in the anterior chamber of the eye leads to intraocular tumor formation. Tumor growth is transient in immunocompetent mice, but lethal in immunodeficient nude mice, indicating that T cell-dependent immunity is responsible for tumor clearance. Tumor rejection has all the characteristics of a CD8(+) T cell-mediated immune response, as the tumor did not express MHC class II and only tumor tissue was the subject of destruction. However, analysis of the molecular and cellular mechanisms involved in tumor clearance revealed that perforin, TNF-alpha, Fas ligand, MHC class I, and CD8(+) T cells did not play a crucial role in tumor eradication. Instead, effective tumor rejection was entirely dependent on CD4(+) Th cells, as CD4-depleted as well as MHC class II-deficient mice were unable to reject their intraocular tumor. Taken together, these observations demonstrate that CD4(+) T cells are able to eradicate MHC class II-negative tumors in an immune-privileged site without affecting surrounding tissues or the induction of phthisis.     

4-1BB costimulation is required for protective anti-viral immunity after peptide vaccination

Peptide vaccination induces T cell activation and cytotoxic T cell development. In an effort to understand what factors can improve immune responses to peptide vaccination, the role of 4-1BB (CD137) costimulation was examined, since 4-1BB has been shown to promote T cell responses in other systems. 4-1BBL-deficient (-/-) and wild-type (+/+) mice were immunized with a lipidated lymphocytic choriomeningitis virus (LCMV) peptide NP396-404. Analysis of peptide-specific responses early after immunization by CTL assay, intracellular IFN-gamma staining, and IFN-gamma enzyme-linked immunospot assay (ELISPOT) indicated that CD8 T cell responses were reduced 3- to 10-fold in the absence of 4-1BB costimulation. Moreover, when agonistic anti-4-1BB Ab was given, CD8 T cell responses in 4-1BBL-/- mice were augmented to levels similar to those in 4-1BBL+/+ mice. Two months after immunization, 4-1BBL+/+ mice still had epitope-specific cells and were protected against viral challenge, demonstrating that peptide vaccination can induce long-term protection. In fact, 70% of CD8 T cells were specific for the immunizing peptide after viral challenge, demonstrating that strong, epitope-specific CD8 T cell responses are generated after peptide vaccination. In contrast, peptide-immunized 4-1BBL-/- mice had fewer epitope-specific cells and were impaired in their ability to resolve the infection. These results show that immunization with a single LCMV peptide provides long term protection against LCMV infection and point to costimulatory molecules such as 4-1BB as important components for generating protective immunity after vaccination.     

The potency and durability of DNA- and protein-based vaccines against Leishmania major evaluated using low-dose, intradermal challenge

DNA- and protein- based vaccines against cutaneous leishmaniasis due to Leishmania major were evaluated using a challenge model that more closely reproduces the pathology and immunity associated with sand fly-transmitted infection. C57BL/6 mice were vaccinated s.c. with a mixture of plasmid DNAs encoding the Leishmania Ags LACK, LmSTI1, and TSA (AgDNA), or with autoclaved L. major promastigotes (ALM) plus rIL-12, and the mice were challenged by inoculation of 100 metacyclic promastigotes in the ear dermis. When challenged at 2 wk postvaccination, mice receiving AgDNA or ALM/rIL-12 were completely protected against the development of dermal lesions, and both groups had a 100-fold reduction in peak dermal parasite loads compared with controls. When challenged at 12 wk, mice vaccinated with ALM/rIL-12 maintained partial protection against dermal lesions and their parasite loads were no longer significantly reduced, whereas the mice vaccinated with AgDNA remained completely protected and had a 1000-fold reduction in dermal parasite loads. Mice vaccinated with AgDNA also harbored few, if any, parasites in the skin during the chronic phase, and their ability to transmit L. major to vector sand flies was completely abrogated. The durable protection in mice vaccinated with AgDNA was associated with the recruitment of both CD8(+) and CD4(+) T cells to the site of intradermal challenge and with IFN-gamma production by CD8(+) T cells in lymph nodes draining the challenge site. These data suggest that under conditions of natural challenge, DNA vaccination has the capacity to confer complete protection against cutaneous leishmaniasis and to prevent the establishment of infection reservoirs.