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.     

Functional CD8+ T cell responses in lethal Ebola virus infection

Ebola virus (EBOV) causes highly lethal hemorrhagic fever that leads to death in up to 90% of infected humans. Like many other infections, EBOV induces massive lymphocyte apoptosis, which is thought to prevent the development of a functional adaptive immune response. In a lethal mouse model of EBOV infection, we show that there is an increase in expression of the activation/maturation marker CD44 in CD4(+) and CD8(+) T cells late in infection, preceding a dramatic rebound of lymphocyte numbers in the blood. Furthermore, we observed both lymphoblasts and apoptotic lymphocytes in spleen late in infection, suggesting that there is lymphocyte activation despite substantial bystander apoptosis. To test whether these activated lymphocytes were functional, we performed adoptive transfer studies. Whole splenocytes from moribund day 7 EBOV-infected animals protected naive animals from EBOV, but not Marburgvirus, challenge. In addition, we observed EBOV-specific CD8(+) T cell IFN-gamma responses in moribund day 7 EBOV-infected mice, and adoptive transfer of CD8(+) T cells alone from day 7 mice could confer protection to EBOV-challenged naive mice. Furthermore, CD8(+) cells from day 7, but not day 0, mice proliferated after transfer to infected recipients. Therefore, despite significant lymphocyte apoptosis, a functional and specific, albeit insufficient, adaptive immune response is made in lethal EBOV infection and is protective upon transfer to naive infected recipients. These findings should cause a change in the current view of the 'impaired' immune response to EBOV challenge and may help spark new therapeutic strategies to control lethal filovirus disease.     

Vaccination against lymphocytic choriomeningitis virus infection in MHC class II-deficient mice

The impact of prophylactic vaccination against acute and chronic infection in a Th-deficient host has not been adequately addressed because of difficulties in generating protective immunity in the absence of CD4(+) T cell help. In this study, we demonstrated that a broad CD8(+) T cell immune response could be elicited in MHC class II-deficient mice by vaccination with adenovirus encoding lymphocytic choriomeningitis virus (LCMV) glycoprotein tethered to MHC class II-associated invariant chain. Moreover, the response induced conferred significant cytolytic CD8(+) T cell-mediated protection against challenge with a high dose of the invasive clone 13 strain of LCMV. In contrast, vaccination with adenovirus encoding unlinked LCMV glycoprotein induced weak virus control in the absence of CD4(+) T cells, and mice may die of increased immunopathology associated with incomplete protection. Acute mortality was not observed in any vaccinated mice following infection with the less-invasive Traub strain. However, LCMV Traub infection caused accelerated late mortality in unvaccinated MHC class II-deficient mice; in this case, we observed a strong trend toward delayed mortality in vaccinated mice, irrespective of the nature of the vaccine. These results indicated that optimized vaccination may lead to efficient protection against acute viral infection, even in Th-deficient individuals, but that the duration of such immunity is limited. Nevertheless, for select immunodeficiencies in which CD4(+) T cell deficiency is incomplete or transient, these results are very encouraging.     

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.     

Protective Mechanisms Induced by a Japanese Encephalitis Virus DNA Vaccine: Requirement for Antibody but Not CD8+ Cytotoxic T-Cell Responses

We have previously shown that a plasmid (pE) encoding the Japanese encephalitis virus (JEV) envelope (E) protein conferred a high level of protection against a lethal viral challenge. In the present study, we used adoptive transfer experiments and gene knockout mice to demonstrate that the DNA-induced E-specific antibody alone can confer protection in the absence of cytotoxic T-lymphocyte (CTL) functions. Plasmid pE administered by either intramuscular or gene gun injection produced significant E-specific antibodies, helper T (Th)-cell proliferative responses, and CTL activities. Animals receiving suboptimal DNA vaccination produced low titers of anti-E antibodies and were only partially or not protected from viral challenge, indicating a strong correlation between anti-E antibodies and the protective capacity. This observation was confirmed by adoptive transfer experiments. Intravenous transfer of E-specific antisera but not crude or T-cell-enriched immune splenocytes to sublethally irradiated hosts conferred protection against a lethal JEV challenge. Furthermore, experiments with gene knockout mice showed that DNA vaccination did not induce anti-E titers and protective immunity in Igmu(-/-) and I-Abeta(-/-) mice, whereas in CD8alpha(-/-) mice the pE-induced antibody titers and protective rate were comparable to those produced in the wild-type mice. Taken together, these results demonstrate that the anti-E antibody is the most critical protective component in this JEV challenge model and that production of anti-E antibody by pE DNA vaccine is dependent on the presence of CD4(+) T cells but independent of CD8(+) T cells.     

CD40 signaling converts a minimally immunogenic antigen into a potent vaccine against the intracellular pathogen Listeria monocytogenes

Conventional vaccination strategies have failed for numerous pathogens, and the development of novel approaches to vaccine development is a major public health priority. Killed or subunit vaccines represent an attractive approach due to their safety, but they suffer from low immunogenicity and generally require adjuvants. In this study, the possibility of harnessing CD40 signaling for enhancing the immunogenicity of killed vaccines was investigated. Intravenous immunization of C57BL/6 mice with heat-killed Listeria monocytogenes (HKL) induced minimal immunity, but HKL administered together with an agonistic anti-CD40 mAb induced high levels of both CD4(+) and CD8(+) T cells capable of producing IFN-gamma following in vitro HKL stimulation. HKL/anti-CD40 vaccination elicited robust protection against subsequent Listeria challenge. Approximately 1000-fold fewer bacteria were detected in the liver and spleen of vaccinated mice, and vaccinated mice were also able to resist a normally lethal Listeria challenge. CD40-mediated adjuvant activity required endogenous IL-12 at the time of vaccination, and protection was mediated by both CD8(+) and CD4(+) T cells. Thus, CD40 signaling can deliver potent adjuvant activity for vaccination against intracellular pathogens and is particularly effective for pathogens requiring both CD4(+) and CD8(+) T cells for effective control.     

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.     

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.     

Depletion of CD4+ T cells during immunization with nonviable Listeria monocytogenes causes enhanced CD8+ T cell-mediated protection against listeriosis

Immunization of mice with nonviable Listeria monocytogenes generates an insufficient CD8(+) T cell response and consequently only limited protection against subsequent L. monocytogenes infection. We have recently demonstrated that depletion of regulatory CD4(+) T cells during immunization significantly enhances CD8(+) T cell responses. In the present study, we determined the impact of CD4(+) T cell depletion on the CD8(+) T cell response against heat-killed LISTERIA: Treatment of mice with anti-CD4 mAb during boost immunization with heat-killed Listeria significantly increased numbers of Listeria-specific CD8(+) T cells and improved protection against subsequent infection with L. monocytogenes. During challenge infection, numbers of Listeria-specific CD8(+) T cells were enhanced, and these cells expressed effector functions in terms of IFN-gamma production. In summary, we demonstrate that combining nonviable L. monocytogenes vaccination and CD4(+) T cell depletion improves generation of long-lasting and functional Listeria-specific CD8(+) memory T cells.     

IL-15 prolongs the duration of CD8+ T cell-mediated immunity in mice infected with a vaccine strain of Toxoplasma gondii.

Immunization of mice with a vaccine (ts-4) strain of Toxoplasma gondii is known to induce complete protection against subsequent lethal infection. Ts-4-mediated protection has been reported to be primarily dependent on IFN-gamma-producing CD8+ T cells. However, duration of CD8+ T cell-mediated immunity in the ts-4-vaccinated animals is not known. In the present study, the kinetics of the CD8+ T cell response in mice immunized with the ts-4 strain of T. gondii was evaluated. Optimal CD8+ T cell immunity persisted at least 6 mo after vaccination, and mice at this time point continued to overcome lethal challenge with a more virulent strain. However, at 9 mo postimmunization, CD8+ T cell immunity was severely diminished and the mice succumbed to Toxoplasma challenge. Pretreatment of animals, vaccinated 9 mo earlier, with rIL-15 prevented the mortality induced by Toxoplasma challenge. The protective effect of IL-15 treatment was due to a rise in the frequency of Ag-specific CD8+ T cells. CD8+ T cells from IL-15-administered animals showed increased proliferation and IFN-gamma production in response to antigenic restimulation. These findings suggest that rIL-15 can reverse the decline in the long-term CD8+ T cell immune response in mice immunized with vaccine strain of T. gondii.     

Induction of HIV-1-specific immunity after vaccination with apoptotic HIV-1/murine leukemia virus-infected cells

Ag-presenting dendritic cells present viral Ags to T cells after uptake of apoptotic bodies derived from virus-infected cells in vitro. However, it is unclear whether apoptotic virus-infected cells are capable of generating immunity in vivo. In this study, we show that inoculation of mice with apoptotic HIV-1/murine leukemia virus (MuLV)-infected cells induces HIV-1-specific immunity. Immunization with apoptotic HIV-1/MuLV-infected syngeneic splenocytes resulted in strong Nef-specific CD8(+) T cell proliferation and p24-induced CD4(+) and CD8(+) T cell proliferation as well as IFN-gamma production. In addition, systemic IgG and IgA as well as mucosa-associated IgA responses were generated. Moreover, mice vaccinated with apoptotic HIV-1/MuLV cells were protected against challenge with live HIV-1/MuLV-infected cells, whereas mice vaccinated with apoptotic noninfected or MuLV-infected splenocytes remained susceptible to HIV-1/MuLV. These data show that i.p. immunization with apoptotic HIV-1-infected cells induces high levels of HIV-1-specific systemic immunity, primes for mucosal immunity, and induces protection against challenge with live HIV-1-infected cells in mice. These findings may have implications for the development of therapeutic and prophylactic HIV-1 vaccines.     

Priming with cold-adapted influenza A does not prevent infection but elicits long-lived protection against supralethal challenge with heterosubtypic virus

We show in this study several novel features of T cell-based heterosubtypic immunity against the influenza A virus in mice. First, T cell-mediated heterosubtypic protection against lethal challenge can be generated by a very low priming dose. Second, it becomes effective within 5-6 days. Third, it provides protection against a very high dose challenge for >70 days. Also novel is the finding that strong, long-lasting, heterosubtypic protection can be elicited by priming with attenuated cold-adapted strains. We demonstrate that priming does not prevent infection of the lungs following challenge, but leads to earlier clearance of the virus and 100% survival after otherwise lethal challenge. Protection is dependent on CD8 T cells, and we show that CD4 and CD8 T cells reactive to conserved epitopes of the core proteins of the challenge virus are present after priming. Our results suggest that intranasal vaccination with cold-adapted, attenuated live virus has the potential to provide effective emergency protection against emerging influenza strains for several months.     

Gamma delta T cells facilitate adaptive immunity against West Nile virus infection in mice

West Nile (WN) virus causes fatal meningoencephalitis in laboratory mice, and gammadelta T cells are involved in the protective immune response against viral challenge. We have now examined whether gammadelta T cells contribute to the development of adaptive immune responses that help control WN virus infection. Approximately 15% of TCRdelta(-/-) mice survived primary infection with WN virus compared with 80-85% of the wild-type mice. These mice were more susceptible to secondary challenge with WN virus than the wild-type mice that survived primary challenge with the virus. Depletion of gammadelta T cells in wild-type mice that survived the primary infection, however, does not affect host susceptibility during secondary challenge with WN virus. Furthermore, gammadelta T cells do not influence the development of Ab responses during primary and at the early stages of secondary infection with WN virus. Adoptive transfer of CD8(+) T cells from wild-type mice that survived primary infection with WN virus to naive mice afforded partial protection from lethal infection. In contrast, transfer of CD8(+) T cells from TCRdelta(-/-) mice that survived primary challenge with WN virus failed to alter infection in naive mice. This difference in survival correlated with the numeric and functional reduction of CD8 memory T cells in these mice. These data demonstrate that gammadelta T cells directly link innate and adaptive immunity during WN virus infection.     

CTL-dependent and -independent antitumor immunity is determined by the tumor not the vaccine

Previously, we compared the efficiency of direct injection with an adenovirus (Ad) expressing human gp100 (hgp100) to immunization with dendritic cells (DC) loaded with the same vector ex vivo. The DC vaccine provided the greatest protection against challenge with B16F10 melanoma, and antitumor immunity was found to be CD8(+) T cell-independent. In the current study, we sought to determine whether lack of CD8(+) T cell-mediated antitumor immunity was a function of the vaccine platform or the tumor line. Both Ad and DC/Ad vaccines elicited CD8(+) CTL reactive against hgp100 and provided protection against B16F10 engineered to express hgp100 demonstrating that both vaccination platforms can effectively generate protective CD8(+) T cell-mediated immunity. The hgp100-induced CTL cross-reacted with murine gp100 (mgp100) and lysed B16F10 cells pulsed with mgp100 peptide indicating that the resistance of B16F10 cells to CTL elicited by hgp100 vaccination may be due to a defect in processing of the endogenous mgp100. Indeed, introduction of the TAP-1 cDNA into B16F10 rendered the cells sensitive to lysis by gp100-specific CTL. Furthermore, gp100-immunized mice were protected from challenge with B16F10-TAP1 cells through a mechanism dependent upon CD8(+) T cells. These results demonstrate that tumor phenotype, not the vaccination platform, ultimately determines CD8(+) or CD4(+) T cell-mediated tumor clearance.     

The protective efficacy of chlamydial protease-like activity factor vaccination is dependent upon CD4+ T cells

We have previously determined the protective efficacy of intranasal vaccination with chlamydial protease-like activity factor (CPAF) against genital chlamydial infection. Since T-helper 1 (Th1) responses are important for anti-chlamydial immunity, we examined the contribution of CD4(+) T cells in CPAF mediated immunity against intravaginal (i.vag.) Chlamydia muridarum infection in C57BL/6 mice. CPAF+IL-12 vaccination induced antigen-specific CD4(+) T cells that secreted elevated levels of IFN-gamma, and generated strong humoral responses. The protective effects of CPAF vaccination against genital chlamydial challenge were abrogated by anti-CD4 neutralizing antibody treatment. Moreover, anti-chlamydial immunity could be adoptively transferred to na?ve recipients using CPAF-specific CD4(+) T cells. Therefore, CPAF mediated anti-chlamydial immunity is highly dependent upon antigen-specific CD4(+) T cells.     

HLA-A2-restricted protection against lethal lymphocytic choriomeningitis

The consequences of human lymphocytic choriomeningitis virus (LCMV) infection can be severe, including aseptic meningitis in immunocompetent individuals, hydrocephalus or chorioretinitis in fetal infection, or a highly lethal outcome in immunosuppressed individuals. In murine models of LCMV infection, CD8(+) T cells play a primary role in providing protective immunity, and there is evidence that cellular immunity may also be important in related arenavirus infections in humans. For this reason, we sought to identify HLA-A2 supertype-restricted epitopes from the LCMV proteome and evaluate them as vaccine determinants in HLA transgenic mice. We identified four HLA-A*0201-restricted peptides-nucleoprotein NP(69-77), glycoprotein precursor GPC(10-18), GPC(447-455), and zinc-binding protein Z(49-58)-that displayed high-affinity binding (< or =275 nM) to HLA-A*0201, induced CD8(+) T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LCMV antigens in HLA-restricted human antigen presenting cells. One of the epitopes (GPC(447-455)), after peptide immunization of HLA-A*0201 mice, induced CD8(+) T cells capable of killing peptide-pulsed HLA-A*0201-restricted target cells in vivo and protected mice against lethal intracranial challenge with LCMV.     

Enhanced tumor responses to dendritic cells in the absence of CD8-positive cells

Wild-type mice immunized with MART-1 melanoma Ag-engineered dendritic cells (DC) generate strong Ag-specific immunity that has an absolute requirement for both CD8(+) and CD4(+) T cells. DC administration to CD8 alpha knockout mice displayed unexpectedly enhanced levels of protection to tumor challenge despite this deficiency in CD8(+) T cells and the inability to mount MHC class I-restricted immune responses. This model has the following features: 1) antitumor protection is Ag independent; 2) had an absolute requirement for CD4(+) and NK1.1(+) cells; 3) CD4(+) splenocytes are responsible for cytokine production; 4) lytic cells in microcytotoxicity assays express NK, but lack T cell markers (NK1.1(+) alpha beta TCR(-) CD3(-)); and 5) the lytic phenotype can be transferred to naive CD8 alpha knockout mice by NK1.1(+) splenocytes. Elucidation of the signaling events that activate these effective cytotoxic cells and the putative suppressive mechanisms in a wild-type environment may provide means to enhance the clinical activity of DC-based approaches.     

Multiple epitopes in the murine cytomegalovirus early gene product M84 are efficiently presented in infected primary macrophages and contribute to strong CD8+-T-lymphocyte responses and protection following DNA immunization

We previously demonstrated that after vaccination of BALB/c mice with DNA encoding murine cytomegalovirus (MCMV) IE1 or M84, a similar level of protection against MCMV infection was achieved. However, the percentage of antigen-specific CD8(+) T cells elicited by IE1 was higher than that by M84 as measured by intracellular cytokine staining when splenocytes were stimulated with an epitope peptide (M. Ye at al., J. Virol. 76:2100-2112, 2002). We show here that after DNA vaccination with M84, a higher percentage of M84-specific CD8(+) T cells was detected when splenocytes were stimulated with J774 cells expressing full-length M84. When the defined M84 epitope 297-305 was deleted, the mutant DNA vaccine was still protective against MCMV replication and induced strong M84-specific CD8(+)-T-cell responses. The M84 gene was subsequently subcloned into three fragments encoding overlapping protein fragments. When mice were immunized with each of the M84 subfragment DNAs, at least two additional protective CD8(+)-T-cell epitopes were detected. In contrast to strong responses after DNA vaccination, M84-specific CD8(+)-T-cell responses were poorly induced during MCMV infection. The weak M84-specific response after MCMV infection was not due to poor antigen presentation in antigen-presenting cells, since both J774 macrophages and primary peritoneal macrophages infected with MCMV in vitro were able to efficiently and constitutively present M84-specific epitopes starting at the early phase of infection. These results indicate that antigen presentation by macrophages is not sufficient for M84-specific CD8(+)-T-cell responses during MCMV infection.     

Class Ia MHC-deficient BALB/c mice generate CD8+ T cell-mediated protective immunity against Listeria monocytogenes infection

CD8(+) T cells are required for protective immunity against intracellular pathogens such as Listeria monocytogenes. In this study, we used class Ia MHC-deficient mice, which have a severe reduction in circulating CD8(+) T cells, to determine the protective capacity of class Ib MHC-restricted T cells during L. monocytogenes infection. The K(b-/-)D(b-/-) mutation was backcrossed onto a C.B10 (BALB/c congenic at H-2 locus with C57BL/10) background, because BALB/c mice are more susceptible to Listeria infection than other commonly studied mouse strains such as C57BL/6. C.B10 K(b-/-)D(b-/-) mice immunized with a sublethal dose of L. monocytogenes were fully protected against a subsequent lethal infection. Adoptive transfer of Listeria-immune splenocyte subsets into naive K(b-/-)D(b-/-) mice indicated that CD8(+) T cells were the major component of this protective immune response. A CD8(+) T cell line isolated from the spleen of a Listeria-infected class Ia MHC-deficient mouse was shown to specifically recognize Listeria-infected cells in vitro, as determined by IFN-gamma secretion and cytotoxicity assays. Adoptive transfer of this T cell line alone resulted in significant protection against L. monocytogenes challenge. These results suggest that even a limited number of class Ib MHC-restricted T cells are sufficient to generate the rapid recall response required for protection against secondary infection with L. monocytogenes.     

Retinoic acid as a vaccine adjuvant enhances CD8+ T cell response and mucosal protection from viral challenge

Vaccine-induced memory T cells localized at mucosal sites can provide rapid protection from viral infection. All-trans-retinoic acid (ATRA) has been shown to act physiologically to induce the expression of gut-homing receptors on lymphocytes. We tested whether the administration of exogenous ATRA during a systemic vaccination of mice could enhance the generation of mucosal CD8(+) T cell immunity, which might represent a strategy for establishing better protection from viral infection via mucosal routes. ATRA induced the expression of CCR9 and α4β7 on both mouse and human CD8(+) T cells activated in vitro. The administration of ATRA to mice during in vivo priming with a replication-defective recombinant adenovirus vector expressing the lymphocytic choriomeningitis virus glycoprotein (LCMVgp) (Ad5gp) increased numbers of both effector and memory T cells in intestinal mucosal tissues and showed higher frequencies of systemic central memory-like T cells that exhibited enhanced proliferation during boosting immunization with recombinant modified vaccinia virus Ankara expressing LCMVgp (MVAgp). Mice that received ATRA during Ad5gp vaccination were more resistant to intravaginal challenge by recombinant vaccinia virus expressing LCMVgp (VVgp), reflecting in part stronger T cell recall responses in situ. Thus, ATRA appears to be useful as an adjuvant during vaccination to increase memory T cell responses and protection from viral infection at mucosal sites and may facilitate the development of more effective vaccines against mucosally transmitted pathogens such as HIV.