T cells as mediators of protective immunity against liver stages of Plasmodium

T cells from different subsets play a major role in protective immunity against pre-erythrocytic stages of malaria parasites. Exposure of humans and animals to malaria sporozoites induces (alphabeta CD8(+) and CD4(+) T cells specific for antigens expressed in pre-erythrocytic stages of Plasmodium. These T cells inhibit parasite development in the liver, and immunization with subunit vaccines expressing the respective antigenic moieties confers protection against sporozoite challenge. gammadelta and natural killer T cells can also play a role in protective immunity. Recent studies with mice transgenic for the alphabeta T-cell receptor have revealed the existence of complex mechanisms regulating the induction and development of these responses.     

Selectively impaired CD8+ but not CD4+ T cell cycle arrest during priming as a consequence of dendritic cell interaction with plasmodium-infected red cells

Individuals living in malaria-endemic areas show generally low T cell responses to malaria Ags. In this study, we show murine dendritic cell (DC) interaction with parasitized erythrocytes (pRBC) arrested their maturation, resulting in impaired ability to stimulate naive, but not recall T cell responses in vitro and in vivo. Moreover, within the naive T cell population, pRBC-treated DC were selectively deficient in priming CD8(+) but not CD4(+) T cells. Indeed, DC that had taken up pRBC were shown for the first time to efficiently prime CD4(+) T cell responses to a known protective merozoite Ag, MSP4/5. In contrast, impaired priming resulted in decreases in both proliferation and cytokine production by CD8(+) T cells. Deficient priming was observed to both a model and a Plasmodium berghei-specific CD8(+) T cell epitope. The mechanisms underlying the inability of parasite-treated DC to prime CD8(+) T cells were explored. pRBC treatment of DC from wild-type C57BL/6, but not from IL-10 knockout animals, suppressed DC-mediated T cell priming across a Transwell, suggesting active IL-10-dependent suppression. CD8(+) T cells were arrested at the G(0) stage of the cell cycle after two cell divisions post-Ag stimulation. The proliferation arrest was partially reversible by the addition of IL-2 or IL-7 to responder cultures. These results suggest that in malaria-endemic areas, priming of CD8(+) T cell responses may be more difficult to induce via vaccination than the priming of CD4(+) T cells. Moreover, pathogens may selectively target the CD8(+) T cell arm of protective immunity for immune evasion.     

IL-12 is required for induction but not maintenance of protective, memory responses to Blastomyces dermatitidis: implications for vaccine development in immune-deficient hosts

Cellular immunity mediated by T lymphocytes, in particular CD4(+) and CD8(+) type 1 (T1) cells, is the main defense against pathogenic fungi. IL-12 initiates T1 cell development and cell-mediated immunity, but it is unclear whether IL-12 contributes to the maintenance of an antifungal T1 response. In this study, we addressed the role of IL-12 for vaccine-induced memory T cell development against experimental pulmonary blastomycosis. CD4(+) T cells absolutely required IL-12 to control a live genetically engineered attenuated strain of Blastomyces dermatitidis given s.c. as a vaccine, whereas CD8(+) T cells were significantly less dependent on IL-12. Despite differential dependency of T cell subsets on IL-12 during vaccination, neither subset acquired memory immunity in the absence of IL-12. In contrast, adoptive transfer of immune CD4 T cells from wild-type mice into IL-12(-/-) mice showed that CD4(+) T1 memory cells sustained a T1 cytokine profile and remained protective over a period of 6 mo posttransfer. Similarly, memory CD8 cells elicited in IL-12(-/-) mice with killed yeast and transient rIL-12 treatment (during vaccination) remained durable and protective after animals were rested for 3 mo. In conclusion, these studies demonstrate that once CD4 and CD8 cells have acquired a protective T1 phenotype they no longer require the presence of IL-12 to maintain antifungal protective memory.     

IL-12 and NK cells are required for antigen-specific adaptive immunity against malaria initiated by CD8+ T cells in the Plasmodium yoelii model.

CD8+ T cells have been implicated as critical effector cells in protection against preerythrocytic stage malaria, including the potent protective immunity of mice and humans induced by immunization with radiation-attenuated Plasmodium spp. sporozoites. This immunity is directed against the Plasmodium spp. parasite developing within the host hepatocyte and for a number of years has been presumed to be mediated directly by CD8+ CTL or indirectly by IFN-gamma released from CD8+ T cells. In this paper, in BALB/c mice, we establish that after immunization with irradiated sporozoites or DNA vaccines parasite-specific CD8+ T cells trigger a novel mechanism of adaptive immunity that is dependent on T cell- and non-T cell-derived cytokines, in particular IFN-gamma and IL-12, and requires NK cells but not CD4+ T cells. The absolute requirement for CD8+ T cells to initiate such an effector mechanism, and the requirement for IL-12 and NK cells in such vaccine-induced protective immunity, are unique and underscore the complexity of the immune responses that protect against malaria and other intracellular pathogens.     

The spleen CD4+ T cell response to blood-stage Plasmodium chabaudi malaria develops in two phases characterized by different properties

The pivotal role of spleen CD4(+) T cells in the development of both malaria pathogenesis and protective immunity makes necessary a profound comprehension of the mechanisms involved in their activation and regulation during Plasmodium infection. Herein, we examined in detail the behaviour of non-conventional and conventional splenic CD4(+) T cells during P. chabaudi malaria. We took advantage of the fact that a great proportion of CD4(+) T cells generated in CD1d(-/-) mice are I-A(b)-restricted (conventional cells), while their counterparts in I-A(b-/-) mice are restricted by CD1d and other class IB major histocompatibility complex (MHC) molecules (non-conventional cells). We found that conventional CD4(+) T cells are the main protagonists of the immune response to infection, which develops in two consecutive phases concomitant with acute and chronic parasitaemias. The early phase of the conventional CD4(+) T cell response is intense and short lasting, rapidly providing large amounts of proinflammatory cytokines and helping follicular and marginal zone B cells to secrete polyclonal immunoglobulin. Both TNF-α and IFN-γ production depend mostly on conventional CD4(+) T cells. IFN-γ is produced simultaneously by non-conventional and conventional CD4(+) T cells. The early phase of the response finishes after a week of infection, with the elimination of a large proportion of CD4(+) T cells, which then gives opportunity to the development of acquired immunity. Unexpectedly, the major contribution of CD1d-restricted CD4(+) T cells occurs at the beginning of the second phase of the response, but not earlier, helping both IFN-γ and parasite-specific antibody production. We concluded that conventional CD4(+) T cells have a central role from the onset of P. chabaudi malaria, acting in parallel with non-conventional CD4(+) T cells as a link between innate and acquired immunity. This study contributes to the understanding of malaria immunology and opens a perspective for future studies designed to decipher the molecular mechanisms behind immune responses to Plasmodium infection.     

Annual vaccination against influenza virus hampers development of virus-specific CD8⁺ T cell immunity in children

Infection with seasonal influenza A viruses induces immunity to potentially pandemic influenza A viruses of other subtypes (heterosubtypic immunity). We recently demonstrated that vaccination against seasonal influenza prevented the induction of heterosubtypic immunity against influenza A/H5N1 virus induced by infection with seasonal influenza in animal models, which correlated with the absence of virus-specific CD8(+) T cell responses. Annual vaccination of all healthy children against influenza has been recommended, but the impact of vaccination on the development of the virus-specific CD8(+) T cell immunity in children is currently unknown. Here we compared the virus-specific CD8(+) T cell immunity in children vaccinated annually with that in unvaccinated children. In the present study, we compared influenza A virus-specific cellular and humoral responses of unvaccinated healthy control children with those of children with cystic fibrosis (CF) who were vaccinated annually. Similar virus-specific CD4(+) T cell and antibody responses were observed, while an age-dependent increase of the virus-specific CD8(+) T cell response that was absent in vaccinated CF children was observed in unvaccinated healthy control children. Our results indicate that annual influenza vaccination is effective against seasonal influenza but hampers the development of virus-specific CD8(+) T cell responses. The consequences of these findings are discussed in the light of the development of protective immunity to seasonal and future pandemic influenza viruses.     

Malaria-specific and nonspecific activation of CD8+ T cells during blood stage of Plasmodium berghei infection

Cerebral malaria is one of the severe complications of Plasmodium falciparum infection. Studies using a rodent model of Plasmodium berghei ANKA infection established that CD8(+) T cells are involved in the pathogenesis of cerebral malaria. However, it is unclear whether and how Plasmodium-specific CD8(+) T cells can be activated during the erythrocyte stage of malaria infection. We generated recombinant Plasmodium berghei ANKA expressing OVA (OVA-PbA) to investigate the parasite-specific T cell responses during malaria infection. Using this model system, we demonstrate two types of CD8(+) T cell activations during the infection with malaria parasite. Ag (OVA)-specific CD8(+) T cells were activated by TAP-dependent cross-presentation during infection with OVA-PbA leading to their expression of an activation phenotype and granzyme B and the development to functional CTL. These highly activated CD8(+) T cells were preferentially sequestered in the brain, although it was unclear whether these cells were involved in the pathogenesis of cerebral malaria. Activation of OVA-specific CD8(+) T cells in RAG2 knockout TCR-transgenic mice during infection with OVA-PbA did not have a protective role but rather was pathogenic to the host as shown by their higher parasitemia and earlier death when compared with RAG2 knockout mice. The OVA-specific CD8(+) T cells, however, were also activated during infection with wild-type parasites in an Ag-nonspecific manner, although the levels of activation were much lower. This nonspecific activation occurred in a TAP-independent manner, appeared to require NK cells, and was not by itself pathogenic to the host.     

Trypanosoma cruzi adjuvants potentiate T cell-mediated immunity induced by a NY-ESO-1 based antitumor vaccine

Immunological adjuvants that induce T cell-mediate immunity (TCMI) with the least side effects are needed for the development of human vaccines. Glycoinositolphospholipids (GIPL) and CpGs oligodeoxynucleotides (CpG ODNs) derived from the protozoa parasite Trypanosoma cruzi induce potent pro-inflammatory reaction through activation of Toll-Like Receptor (TLR)4 and TLR9, respectively. Here, using mouse models, we tested the T. cruzi derived TLR agonists as immunological adjuvants in an antitumor vaccine. For comparison, we used well-established TLR agonists, such as the bacterial derived monophosphoryl lipid A (MPL), lipopeptide (Pam3Cys), and CpG ODN. All tested TLR agonists were comparable to induce antibody responses, whereas significant differences were noticed in their ability to elicit CD4(+) T and CD8(+) T cell responses. In particular, both GIPLs (GTH, and GY) and CpG ODNs (B344, B297 and B128) derived from T. cruzi elicited interferon-gamma (IFN-γ) production by CD4(+) T cells. On the other hand, the parasite derived CpG ODNs, but not GIPLs, elicited a potent IFN-γ response by CD8(+) T lymphocytes. The side effects were also evaluated by local pain (hypernociception). The intensity of hypernociception induced by vaccination was alleviated by administration of an analgesic drug without affecting protective immunity. Finally, the level of protective immunity against the NY-ESO-1 expressing melanoma was associated with the magnitude of both CD4(+) T and CD8(+) T cell responses elicited by a specific immunological adjuvant.     

Vaccination with lipid core peptides fails to induce epitope-specific T cell responses but confers non-specific protective immunity in a malaria model

Vaccines against many pathogens for which conventional approaches have failed remain an unmet public health priority. Synthetic peptide-based vaccines offer an attractive alternative to whole protein and whole organism vaccines, particularly for complex pathogens that cause chronic infection. Previously, we have reported a promising lipid core peptide (LCP) vaccine delivery system that incorporates the antigen, carrier, and adjuvant in a single molecular entity. LCP vaccines have been used to deliver several peptide subunit-based vaccine candidates and induced high titre functional antibodies and protected against Group A streptococcus in mice. Herein, we have evaluated whether LCP constructs incorporating defined CD4(+) and/or CD8(+) T cell epitopes could induce epitope-specific T cell responses and protect against pathogen challenge in a rodent malaria model. We show that LCP vaccines failed to induce an expansion of antigen-specific CD8(+) T cells following primary immunization or by boosting. We further demonstrated that the LCP vaccines induced a non-specific type 2 polarized cytokine response, rather than an epitope-specific canonical CD8(+) T cell type 1 response. Cytotoxic responses of unknown specificity were also induced. These non-specific responses were able to protect against parasite challenge. These data demonstrate that vaccination with lipid core peptides fails to induce canonical epitope-specific T cell responses, at least in our rodent model, but can nonetheless confer non-specific protective immunity against Plasmodium parasite challenge.     

Enhanced CD8+ T cell immune responses and protection elicited against Plasmodium berghei malaria by prime boost immunization regimens using a novel attenuated fowlpox virus

Sterile immunity can be provided against the pre-erythrocytic stages of malaria by IFN-gamma-secreting CD8(+) T cells that recognize parasite-infected hepatocytes. In this study, we have investigated the use of attenuated fowlpox virus (FPV) strains as recombinant vaccine vectors for eliciting CD8(+) T cells against Plasmodium berghei. The gene encoding the P. berghei circumsporozoite (PbCS) protein was inserted into an FPV vaccine strain licensed for use in chickens, Webster's FPV, and the novel FPV vaccine strain FP9 by homologous recombination. The novel FP9 strain proved more potent as a vaccine for eliciting CD8(+) T cell responses against the PbCS Ag. Sequential immunization with rFP9 and recombinant modified vaccinia virus Anakara (MVA) encoding the PbCS protein, administered by clinically acceptable routes, elicited potent CD8(+) T cell responses against the PbCS protein. This immunization regimen elicited substantial protection against a stringent liver-stage challenge with P. berghei and was more immunogenic and protective than DNA/MVA prime/boost immunization. However, further improvement was not achieved by sequential (triple) immunization with a DNA vaccine, FP9, and MVA.     

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.     

Recombinant viral-vectored vaccines expressing Plasmodium chabaudi AS apical membrane antigen 1: mechanisms of vaccine-induced blood-stage protection

Apical membrane Ag 1 (AMA1) is one of the leading candidate Ags for inclusion in a subunit vaccine against blood-stage malaria. However, the efficacy of Ab-inducing recombinant AMA1 protein vaccines in phase IIa/b clinical trials remains disappointing. In this article, we describe the development of recombinant human adenovirus serotype 5 and modified vaccinia virus Ankara vectors encoding AMA1 from the Plasmodium chabaudi chabaudi strain AS. These vectors, when used in a heterologous prime-boost regimen in BALB/c mice, are capable of inducing strong transgene-specific humoral and cellular immune responses. We show that this vaccination regimen is protective against a nonlethal P. chabaudi chabaudi strain AS blood-stage challenge, resulting in reduced peak parasitemias. The role of vaccine-induced, AMA1-specific Abs and T cells in mediating the antiparasite effect was investigated by in vivo depletion of CD4(+) T cells and adoptive-transfer studies into naive and immunodeficient mice. Depletion of CD4(+) T cells led to a loss of vaccine-induced protection. Adoptive-transfer studies confirmed that efficacy is mediated by both CD4(+) T cells and Abs functioning in the context of an intact immune system. Unlike previous studies, these results confirm that Ag-specific CD4(+) T cells, induced by a clinically relevant vaccine-delivery platform, can make a significant contribution to vaccine blood-stage efficacy in the P. chabaudi model. Given that cell-mediated immunity may also contribute to parasite control in human malaria, these data support the clinical development of viral-vectored vaccines that induce both T cell and Abs against Plasmodium falciparum blood-stage malaria Ags like AMA1.     

No intrinsic deficiencies in CD8+ T cell-mediated antitumor immunity with aging

Aging is associated with a decline in immune function, particularly within the T cell compartment. Because CD8(+) T cells are critical mediators of protective immunity against cancer, which arises more frequently with advancing age, it is important to understand how aging affects T cell-based antitumor responses. We used our DUC18 T cell/CMS5 tumor model system to examine the ability of both aged APCs and aged, tumor-specific CD8(+) T cells to mount protective responses to tumors in vivo. An assessment of aged DUC18 T cells in vitro showed a naive phenotype, but impaired proliferation in response to anti-CD3 and anti-CD28 stimulation. We found that DCs from young and old recipient mice are comparable phenotypically, and endogenous APCs in these mice are equally able to prime adoptively transferred young DUC18 T cells. Even when aged DUC18 T cells are transferred into aged CMS5-challenged mice, Ag-specific proliferation and CD25 expression are similar to those found when young DUC18 T cells are transferred into young mice. Although trafficking to tumor sites appears unequal, old and young DUC18 T cells reject primary CMS5 challenges to the same degree and with similar kinetics. Overall, we found no loss of endogenous APC function or intrinsic defects in CD8(+) DUC18 T cells with advanced age. Therefore, when young and old tumor-specific T cell populations are equivalently sized, CD8(+) T cell-mediated antitumor immunity in our system is not impaired by age, a finding that has positive implications for T cell-based immunotherapies.     

Influence of CD4+CD25+ T cells on Plasmodium berghei NK65 infection in BALB/c mice

CD4(+) T cells co-expressing CD25 (CD4(+)CD25(+) T cells) have been identified as immunoregulatory suppressors modulating autoimmune response. Beside that, autoimmune response was supposed to be associated with malaria infection. Based on these data, we hypothesised that CD4(+)CD25(+) T cells may influence protective immunity to malaria parasites, while suppressing autoimmune response arising throughout the course of malarial infection. To test this possibility, we evaluated the kinetics of CD4(+)CD25(+) T cells during malaria infection and investigated the influence of CD25 depletion by anti-mouse CD25 monoclonal antibody (PC61) on the infection, using a mouse model of premunition to Plasmodium berghei NK65 malaria. The results showed that, during exacerbation of P. berghei NK65 infection, the proportion of CD4(+)CD25(+) T cells among CD4(+) T cells decreased, although that of CD4(+) T cells increased. CD25 depletion clearly delayed the growth of parasitaemia during parasite challenge, particularly in immunised mice. These findings demonstrated that CD4(+)CD25(+) T cells are able to influence protective immunity underlying premunition to P. berghei NK65 parasites.     

The complexity of protective immunity against liver-stage malaria

Sterile protective immunity against challenge with Plasmodium spp. sporozoites can be induced in multiple model systems and humans by immunization with radiation-attenuated Plasmodium spp. sporozoites. The infected hepatocyte has been established as the primary target of this protection, but the underlying mechanisms have not been completely defined. Abs, CD8+ T cells, CD4+ T cells, cytokines (including IFN-gamma and IL-12), and NO have all been implicated as critical effectors. Here, we have investigated the mechanisms of protective immunity induced by immunization with different vaccine delivery systems (irradiated sporozoites, plasmid DNA, synthetic peptide/adjuvant, and multiple Ag peptide) in genetically distinct inbred strains, genetically modified mice, and outbred mice. We establish that there is a marked diversity of T cell-dependent immune responses that mediate sterile protective immunity against liver-stage malaria. Furthermore, we demonstrate that distinct mechanisms of protection are induced in different strains of inbred mice by a single method of immunization, and in the same strain by different methods of immunization. These data underscore the complexity of the murine host response to a parasitic infection and suggest that an outbred human population may behave similarly. Data nevertheless suggest that a pre-erythrocytic-stage vaccine should be designed to induce CD8+ T cell- and IFN-gamma-mediated immune responses and that IFN-gamma responses may represent an in vitro correlate of pre-erythrocytic-stage protective immunity.     

Impaired protection against Mycobacterium bovis bacillus Calmette-Guerin infection in IL-15-deficient mice

To investigate the potential role of endogenous IL-15 in mycobacterial infection, we examined protective immunity in IL-15-deficient (IL-15(-/-)) mice after infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG) or recombinant OVA-expressing BCG (rBCG-OVA). IL-15(-/-) mice exhibited an impaired protection in the lung on day 120 after BCG infection as assessed by bacterial growth. CD4(+) Th1 response capable of producing IFN-gamma was normally detected in spleen and lung of IL-15(-/-) mice on day 120 after infection. Although Ag-specific CD8 responses capable of producing IFN-gamma and exhibiting cytotoxic activity were detected in the lung on day 21 after infection with rBCG-OVA, the responses were severely impaired on days 70 and 120 in IL-15(-/-) mice. The degree of proliferation of Ag-specific CD8(+) T cells in IL-15(-/-) mice was similar to that in wild-type mice during the course of infection with rBCG-OVA, whereas sensitivity to apoptosis of Ag-specific CD8(+) T cells significantly increased in IL-15(-/-) mice. These results suggest that IL-15 plays an important role in the development of long-lasting protective immunity to BCG infection via sustaining CD8 responses in the lung.     

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.     

Stimulation of the glucocorticoid-induced TNF receptor family-related receptor on CD8 T cells induces protective and high-avidity T cell responses to tumor-specific antigens

Treatment of tumor-bearing mice with a stimulatory Ab to glucocorticoid-induced TNFR family-related receptor (GITR) has previously been shown to elicit protective T cell responses against poorly immunogenic tumors. However, the role of GITR stimulation on CD8 T cells and the nature of tumor rejection Ags have yet to be determined. In this study, we show that a stimulatory mAb to GITR (clone DTA-1) acts directly on CD8 T cells, but not on CD4(+)CD25(+) regulatory T (T(reg)) cells, in B16 tumor-bearing mice to induce concomitant immunity against secondary B16 tumors, as well as protective memory following surgical excision of the primary tumor. Melanoma growth itself induced GITR expression on tumor-specific CD8 T cells, providing a mechanism whereby these cells may respond to stimulatory anti-GITR. Unexpectedly, in contrast to T(reg) cell depletion therapy with anti-CD4, GITR stimulation induced very weak CD8 T cell responses to melanocyte differentiation Ags expressed by the tumor, and did not induce autoimmune vitiligo. Accordingly, GITR-stimulated hosts that were primed with B16 melanoma rejected B16, but not the unrelated JBRH melanoma, indicating that tumor rejection Ags are tumor-specific rather than shared. In support of this, we show that GITR stimulation induces CD8 T cell responses to a tumor-specific Ag, and that these responses are of higher functional avidity compared with those induced by T(reg) cell depletion. We conclude that stimulation of GITR on effector CD8 T cells results in high-avidity T cell responses to tumor-specific Ags, thereby inducing potent antitumor immunity in the absence of autoimmunity.     

Immune responses following neonatal DNA vaccination are long-lived, abundant, and qualitatively similar to those induced by conventional immunization

Virus infections are devastating to neonates, and the induction of active antiviral immunity in this age group is an important goal. Here, we show that a single neonatal DNA vaccination induces cellular and humoral immune responses which are maintained for a significant part of the animal's life span. We employ a sensitive technique which permits the first demonstration and quantitation, directly ex vivo, of virus-specific CD8(+) T cells induced by DNA immunization. One year postvaccination, antigen-specific CD8(+) T cells were readily detectable and constituted 0.5 to 1% of all CD8(+) T cells. By several criteria-including cytokine production, perforin content, development of lytic ability, and protective capacity-DNA vaccine-induced CD8(+) memory T cells were indistinguishable from memory cells induced by immunization with a conventional (live-virus) vaccine. Analyses of long-term humoral immune responses revealed that, in contrast to the strong immunoglobulin G2a (IgG2a) skewing of the humoral response seen after conventional vaccination, IgG1 and IgG2a levels were similar in DNA-vaccinated neonatal and adult animals, indicating a balanced T helper response. Collectively, these results show that a single DNA vaccination within hours or days of birth can induce long-lasting CD8(+) T- and B-cell responses; there is no need for secondary immunization (boosting). Furthermore, the observed immune responses induced in neonates and in adults are indistinguishable by several criteria, including protection against virus challenge.     

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.