Neonates mount robust and protective adult-like CD8(+)-T-cell responses to DNA vaccines

Neonates are thought to mount less vigorous adaptive immune responses than adults to antigens and infectious agents. This concept has led to a delay in the administration of many currently available vaccines until late infancy or early childhood. It has recently been shown that vaccines composed of plasmid DNA can induce both humoral and cell-mediated antimicrobial immunity when administered within hours of birth. In most of these studies, immune responses were measured weeks or months after the initial vaccination, and it is therefore questionable whether the observed responses were actually the result of priming of splenocytes within the neonatal period. Here we show that DNA vaccination at birth results in the rapid induction of antigen-specific CD8(+) T cells within neonatal life. Analyses of T-cell effector functions critical for the resolution of many viral infections revealed that neonatal and adult CD8(+) T cells produce similar arrays of cytokines. Furthermore, the avidities of neonatal and adult CD8(+) T cells for peptide and the rapidity with which they upregulate cytokine production after recall encounters with antigen are similar. Protective immunity against the arenavirus lymphocytic choriomeningitis virus, which is mediated by CD8(+) cytotoxic T cells, is also rapidly acquired within the neonatal period. Collectively these data imply that, at least in the case of CD8(+) T cells, neonates are not as immunodeficient as previously supposed and that DNA vaccines may be an effective and safe means of providing critical cell-mediated antiviral immunity extremely early in life.     

HIV-DNA priming alters T cell responses to HIV-adenovirus vaccine even when responses to DNA are undetectable

Many candidate HIV vaccines are designed to primarily elicit T cell responses. Although repeated immunization with the same vaccine boosts Ab responses, the benefit for T cell responses is ill defined. We compared two immunization regimens that include the same recombinant adenoviral serotype 5 (rAd5) boost. Repeated homologous rAd5 immunization fails to increase T cell responses, but increases gp140 Ab responses 10-fold. DNA prime, as compared with rAd5 prime, directs long-term memory CD8(+) T cells toward a terminally differentiated effector memory phenotype with cytotoxic potential. Based on the kinetics of activated cells measured directly ex vivo, the DNA vaccination primes for both CD4(+) and CD8(+) T cells, despite the lack of detection of the latter until after the boost. These results suggest that heterologous prime-boost combinations have distinct immunological advantages over homologous prime-boosts and suggest that the effect of DNA on subsequent boosting may not be easily detectable directly after the DNA vaccination.     

High quality long-term CD4+ and CD8+ effector memory populations stimulated by DNA-LACK/MVA-LACK regimen in Leishmania major BALB/c model of infection

Heterologous vaccination based on priming with a plasmid DNA vector and boosting with an attenuated vaccinia virus MVA recombinant, with both vectors expressing the Leishmania infantum LACK antigen (DNA-LACK and MVA-LACK), has shown efficacy conferring protection in murine and canine models against cutaneus and visceral leishmaniasis, but the immune parameters of protection remain ill defined. Here we performed by flow cytometry an in depth analysis of the T cell populations induced in BALB/c mice during the vaccination protocol DNA-LACK/MVA-LACK, as well as after challenge with L. major parasites. In the adaptive response, there is a polyfunctional CD4(+) and CD8(+) T cell activation against LACK antigen. At the memory phase the heterologous vaccination induces high quality LACK-specific long-term CD4(+) and CD8(+) effector memory cells. After parasite challenge, there is a moderate boosting of LACK-specific CD4(+) and CD8(+) T cells. Anti-vector responses were largely CD8(+)-mediated. The immune parameters induced against LACK and triggered by the combined vaccination DNA/MVA protocol, like polyfunctionality of CD4(+) and CD8(+) T cells with an effector phenotype, could be relevant in protection against leishmaniasis.     

B lymphocytes participate in cross-presentation of antigen following gene gun vaccination

Although endocytosed proteins are commonly presented via the class II MHC pathway to stimulate CD4(+) T cells, professional APCs can also cross-present Ags, whereby these exogenous peptides can be complexed with class I MHC for cross-priming of CD8(+) T cells. Whereas the ability of dendritic cells (DCs) to cross-present Ags is well documented, it is not known whether other APCs may also play a role, or what is the relative contribution of cross-priming to the induction of acquired immunity after DNA immunization. In this study, we compared immune responses generated after gene gun vaccination of mice with DNA vaccine plasmids driven by the conventional CMV promoter, the DC-specific CD11c promoter, or the keratinocyte-specific K14 promoter. The CD11c promoter achieved equivalent expression in CD11c(+) DCs in draining lymph nodes over time, as did a conventional CMV-driven plasmid. However, immunization with DC-restricted DNA vaccines failed to generate protective humoral or cellular immunity to model Ags influenza hemagglutinin and OVA, despite the ability of CD11c(+) cells isolated from lymph nodes to stimulate proliferation of Ag-specific T cells directly ex vivo. In contrast, keratinocyte-restricted vaccines elicited comparable T and B cell activity as conventional CMV promoter-driven vaccines, indicating that cross-priming plays a major role in the generation of immune responses after gene gun immunization. Furthermore, parallel studies in B cell-deficient mu-MT mice demonstrated that B lymphocytes, in addition to DCs, mediate cross-priming of Ag-specific T cells. Collectively, these data indicate that broad expression of the immunogen is required for optimal induction of protective acquired immunity.     

Acellular pertussis booster in adolescents induces Th1 and memory CD8+ T cell immune response

In a number of countries, whole cell pertussis vaccines (wcP) were replaced by acellular vaccines (aP) due to an improved reactogenicity profile. Pertussis immunization leads to specific antibody production with the help of CD4(+) T cells. In earlier studies in infants and young children, wcP vaccines selectively induced a Th1 dominated immune response, whereas aP vaccines led to a Th2 biased response. To obtain data on Th1 or Th2 dominance of the immune response in adolescents receiving an aP booster immunization after a wcP or aP primary immunization, we analyzed the concentration of Th1 (IL-2, TNF-α, INF-γ) and Th2 (IL-4, IL-5, IL-10) cytokines in supernatants of lymphocyte cultures specifically stimulated with pertussis antigens. We also investigated the presence of cytotoxic T cell responses against the facultative intracellular bacterium Bordetella pertussis by quantifying pertussis-specific CD8(+) T cell activation following the aP booster immunization. Here we show that the adolescent aP booster vaccination predominantly leads to a Th1 immune response based on IFNgamma secretion upon stimulation with pertussis antigen, irrespective of a prior whole cell or acellular primary vaccination. The vaccination also induces an increase in peripheral CD8(+)CD69(+) activated pertussis-specific memory T cells four weeks after vaccination. The Th1 bias of this immune response could play a role for the decreased local reactogenicity of this adolescent aP booster immunization when compared to the preceding childhood acellular pertussis booster. Pertussis-specific CD8(+) memory T cells may contribute to protection against clinical pertussis.     

The adjuvant effects of co-stimulatory molecules on cellular and memory responses to HBsAg DNA vaccination

Because DNA vaccines on their own tend to induce weak immune responses in humans, adjuvant methods are needed in order to improve their efficacy. The co-stimulatory molecules 4-1BBL, OX40L, and CD70 have been shown to induce strong T cell activities; therefore, in this study, we investigated whether they may be used as molecular adjuvants for a hepatitis B surface antigen (HBsAg) DNA vaccine (pcDS2) in eliciting strong cellular and memory responses. Compared to mice immunized with pcDS2 alone, addition of the co-stimulatory molecules increased T cell proliferation and an HBsAg-specific antibody response that was marked with a higher ratio of IgG2a/IgG1. Importantly, pcDS2 plus these co-stimulatory molecules elicited a higher level of IFN-gamma and IL-4 in CD4(+) T cells and a higher level of IFN-gamma in CD8(+) T cells. In addition, a significantly robust antigen-specific cytotoxic T lymphocyte (CTL) response and the production of long-term memory CD8(+) T cells were also observed in the groups immunized with pcDS2 plus 4-1BBL, OX40L, or CD70. Consistently, as late as 100 days after immunization, upregulated expressions of BCL-2, Spi2A, IL-7Ra, and IL-15Ra were still observed in mice immunized with pcDS2 plus these co-stimulatory molecules, suggesting the generation of memory T cells in these groups. Together, these results suggest that the co-stimulatory molecules 4-1BBL, OX40L, or CD70 can enhance the immunogenicity of HBsAg DNA vaccines, resulting in strong humoral, cellular, and memory responses. This approach may lead to an effective therapeutic vaccine for chronic hepatitis B virus (HBV) infection.     

Effects of chronic heat stress on immune responses of the foot-and-mouth disease DNA vaccination

The main purpose of this study was to assess the effects of chronic heat stress (CHS) on humoral and cellular responses of DNA vaccination. Mice with the CHS were exposed to a temperature set at 38 +/- 1 degrees C, 2h per day, for 35 days, and mice with thermoneutral (TN) temperature were maintained at 24 +/- 1 degrees C for the same period of time. Both groups of mice were immunized with a DNA vaccine-expressed viruscapsid protein 1 (VP1) of foot-and-mouth disease virus (FMDV), and we tested their antigen-specific humoral and cellular responses during the treatments. Compared with the TN group, titers of total Imunoglobulin G (IgG) and IgG1 and expression of interleukin 4 (IL-4) in CD4(+) cells of CHS group were not affected significantly. In contrast, the levels of IgG2a, T cell proliferations, and expression of interferon-gama (IFN-gamma) in both CD4(+) and CD8(+) cells were suppressed significantly, and cytotoxic T-lymphocyte (CTL) responses in vivo were also weakened by the CHS condition. These results indicate that the CHS treatment has negatively affected the immune responses of DNA vaccination and particularly impaired to the cell-mediated responses. It suggests that vaccination in animals is affected by the changes of ambient temperature.     

Trafficking of antigen-specific CD8+ T lymphocytes to mucosal surfaces following intramuscular vaccination

A critical goal of vaccine development for a wide variety of pathogens is the induction of potent and durable mucosal immunity. However, it has been assumed that this goal would be difficult to achieve by systemic vaccination due to the anatomic and functional distinctness of the systemic and mucosal immune systems and the resultant compartmentalization of immune responses. In this study, we show that Ag-specific CD8(+) T lymphocytes traffic efficiently to mucosal surfaces following systemic vaccination. Intramuscular immunization with recombinant adenovirus (rAd) vector-based vaccines expressing SIV Gag resulted in potent, durable, and functional CD8(+) T lymphocyte responses at multiple mucosal effector sites in both mice and rhesus monkeys. In adoptive transfer studies in mice, vaccine-elicited systemic CD8(+) T lymphocytes exhibited phenotypic plasticity, up-regulated mucosal homing integrins and chemokine receptors, and trafficked rapidly to mucosal surfaces. Moreover, the migration of systemic CD8(+) T lymphocytes to mucosal compartments accounted for the vast majority of Ag-specific mucosal CD8(+) T lymphocytes induced by systemic vaccination. Thus, i.m. vaccination can overcome immune compartmentalization and generate robust mucosal CD8(+) T lymphocyte memory. These data demonstrate that the systemic and mucosal immune systems are highly coordinated following vaccination.     

Maternal LAMP/p55gagHIV-1 DNA immunization induces in utero priming and a long-lasting immune response in vaccinated neonates

Infants born to HIV-infected mothers are at high risk of becoming infected during gestation or the breastfeeding period. A search is thus warranted for vaccine formulations that will prevent mother-to-child HIV transmission. The LAMP/gag DNA chimeric vaccine encodes the HIV-1 p55gag fused to the lysosome-associated membrane protein-1 (LAMP-1) and has been shown to enhance anti-Gag antibody (Ab) and cellular immune responses in adult and neonatal mice; such a vaccine represents a new concept in antigen presentation. In this study, we evaluated the effect of LAMP/gag DNA immunization on neonates either before conception or during pregnancy. LAMP/gag immunization of BALB/c mice before conception by the intradermal route led to the transfer of anti-Gag IgG1 Ab through the placenta and via breastfeeding. Furthermore, there were an increased percentage of CD4+CD25+Foxp3+T cells in the spleens of neonates. When offspring were immunized with LAMP/gag DNA, the anti-Gag Ab response and the Gag-specific IFN-γ-secreting cells were decreased. Inhibition of anti-Gag Ab production and cellular responses were not observed six months after immunization, indicating that maternal immunization did not interfere with the long-lasting memory response in offspring. Injection of purified IgG in conjunction with LAMP/gag DNA immunization decreased humoral and cytotoxic T-cell responses. LAMP/gag DNA immunization by intradermal injection prior to conception promoted the transfer of Ab, leading to a diminished response to Gag without interfering with the development of anti-Gag T- and B-cell memory. Finally, we assessed responses after one intravenous injection of LAMP/gag DNA during the last five days of pregnancy. The intravenous injection led to in utero immunization. In conclusion, DNA vaccine enconding LAMP-1 with Gag and other HIV-1 antigens should be considered in the development of a protective vaccine for the maternal/fetal and newborn periods.     

Enhancing blood-stage malaria subunit vaccine immunogenicity in rhesus macaques by combining adenovirus, poxvirus, and protein-in-adjuvant vaccines

Protein-in-adjuvant formulations and viral-vectored vaccines encoding blood-stage malaria Ags have shown efficacy in rodent malaria models and in vitro assays against Plasmodium falciparum. Abs and CD4(+) T cell responses are associated with protective efficacy against blood-stage malaria, whereas CD8(+) T cells against some classical blood-stage Ags can also have a protective effect against liver-stage parasites. No subunit vaccine strategy alone has generated demonstrable high-level efficacy against blood-stage infection in clinical trials. The induction of high-level Ab responses, as well as potent T and B cell effector and memory populations, is likely to be essential to achieve immediate and sustained protective efficacy in humans. This study describes in detail the immunogenicity of vaccines against P. falciparum apical membrane Ag 1 in rhesus macaques (Macaca mulatta), including the chimpanzee adenovirus 63 (AdCh63), the poxvirus modified vaccinia virus Ankara (MVA), and protein vaccines formulated in Alhydrogel or CoVaccine HT adjuvants. AdCh63-MVA heterologous prime-boost immunization induces strong and long-lasting multifunctional CD8(+) and CD4(+) T cell responses that exhibit a central memory-like phenotype. Three-shot (AdCh63-MVA-protein) or two-shot (AdCh63-protein) regimens induce memory B cells and high-titer functional IgG responses that inhibit the growth of two divergent strains of P. falciparum in vitro. Prior immunization with adenoviral vectors of alternative human or simian serotype does not affect the immunogenicity of the AdCh63 apical membrane Ag 1 vaccine. These data encourage the further clinical development and coadministration of protein and viral vector vaccine platforms in an attempt to induce broad cellular and humoral immune responses against blood-stage malaria Ags in humans.     

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.     

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.     

Prime-boost vaccination with HIV-1 Gag protein and cytosine phosphate guanosine oligodeoxynucleotide,followed by adenovirus,induces sustained and robust humoral and cellular immune responses

A prophylactic vaccine for HIV-1 will probably require the induction and maintenance of both humoral and cellular immunity. One current strategy to achieve such long term immune responses is a prime-boost vaccination approach using a DNA priming inoculation, followed by recombinant viral boost. In this report we use a novel prime-boost approach in which the priming injections consist of recombinant HIV-1 Gag protein mixed with cytosine phosphate guanosine oligodeoxynucleotide (CpG ODN), followed by recombinant adenoviral boost expressing HIV-1 Gag. Analysis of the immune responses indicates that HIV-1 Gag protein plus CpG ODN immunization alone induces potent humoral as well as Th1 and CD8+ T cell responses. Boosting with recombinant adenovirus strikingly enhances CD8+, but not Th1, T cell responses, resulting in CD8+ T cell responses far greater in magnitude than Th1 responses. Furthermore, the Th1 and CD8+ T cell responses following prime-boost immunization were seen in both lymphoid and peripheral mucosal organs and were sustained over several months. Together, these data suggest a new immunization approach for elicitation of long term humoral and cellular immune responses.     

Analysis of immune responses against nucleocapsid protein of the Hantaan virus elicited by virus infection or DNA vaccination

Even though neutralizing antibodies against the Hantaan virus (HTNV) has been proven to be critical against viral infections, the cellular immune responses to HTNV are also assumed to be important for viral clearance. In this report, we have examined the cellular and humoral immune responses against the HTNV nucleocapsid protein (NP) elicited by virus infection or DNA vaccination. To examine the cellular immune response against HTNV NP, we used H-2K(b) restricted T-cell epitopes of NP. The NP-specific CD8(+) T cell response was analyzed using a (51)Cr-release assay, intracellular cytokine staining assay, enzyme-linked immunospot assay and tetramer binding assay in C57BL/6 mice infected with HTNV. Using these methods, we found that HTNV infection elicited a strong NP-specific CD8(+) T cell response at eight days after infection. We also found that several different methods to check the NP-specific CD8(+) T cell response showed a very high correlation among analysis. In the case of DNA vaccination by plasmid encoding nucleocapsid gene, the NP-specific antibody response was elicited 2 approximately 4 weeks after immunization and maximized at 6 approximately 8 weeks. NP-specific CD8(+) T cell response reached its peak 3 weeks after immunization. In a challenge test with the recombinant vaccinia virus expressing NP (rVV-HTNV-N), the rVV-HTNV-N titers in DNA vaccinated mice were decreased about 100-fold compared to the negative control mice.     

Protection from bacterial infection by a single vaccination with replication-deficient mutant herpes simplex virus type 1

Adaptive immune responses in which CD8(+) T cells recognize pathogen-derived peptides in the context of major histocompatibility complex class I molecules play a major role in the host defense against infection with intracellular pathogens. Cells infected with intracellular bacteria such as Listeria monocytogenes, Salmonella enterica serovar Typhimurium, or Mycobacterium tuberculosis are directly lysed by cytotoxic CD8(+) T cells. For this reason, current vaccines for intracellular pathogens, such as subunit vaccines or viable bacterial vaccines, aim to generate robust cytotoxic T-cell responses. In order to investigate the capacity of a herpes simplex virus type 1 (HSV-1) vector to induce strong cytotoxic effector cell responses and protection from infection with intracellular pathogens, we developed a replication-deficient, recombinant HSV-1 (rHSV-1) vaccine. We demonstrate in side-by-side comparison with DNA vaccination that rHSV-1 vaccination induces very strong CD8(+) effector T-cell responses. While both vaccines provided protection from infection with L. monocytogenes at low, but lethal doses, only rHSV-1 vaccines could protect from higher infectious doses; HSV-1 induced potent memory cytotoxic T lymphocytes that, upon challenge by pathogens, efficiently protected the animals. Despite the stimulation of relatively low humoral and CD4-T-cell responses, rHSV-1 vectors are strong candidates for future vaccine strategies that confer efficient protection from subsequent infection with intracellular bacteria.     

Murine polyomavirus virus-like particles carrying full-length human PSA protect BALB/c mice from outgrowth of a PSA expressing tumor

Virus-like particles (VLPs) consist of capsid proteins from viruses and have been shown to be usable as carriers of protein and peptide antigens for immune therapy. In this study, we have produced and assayed murine polyomavirus (MPyV) VLPs carrying the entire human Prostate Specific Antigen (PSA) (PSA-MPyVLPs) for their potential use for immune therapy in a mouse model system. BALB/c mice immunized with PSA-MPyVLPs were only marginally protected against outgrowth of a PSA-expressing tumor. To improve protection, PSA-MPyVLPs were co-injected with adjuvant CpG, either alone or loaded onto murine dendritic cells (DCs). Immunization with PSA-MPyVLPs loaded onto DCs in the presence of CpG was shown to efficiently protect mice from tumor outgrowth. In addition, cellular and humoral immune responses after immunization were examined. PSA-specific CD4(+) and CD8(+) cells were demonstrated, but no PSA-specific IgG antibodies. Vaccination with DCs loaded with PSA-MPyVLPs induced an eight-fold lower titre of anti-VLP antibodies than vaccination with PSA-MPyVLPs alone. In conclusion, immunization of BALB/c mice with PSA-MPyVLPs, loaded onto DCs and co-injected with CpG, induces an efficient PSA-specific tumor protective immune response, including both CD4(+) and CD8(+) cells with a low induction of anti-VLP antibodies.     

CD4+ CD25+ T cells regulate vaccine-generated primary and memory CD8+ T-cell responses against herpes simplex virus type 1

It has become evident that naturally occurring CD25(+) regulatory T cells (T(reg) cells) not only influence self-antigen specific immune response but also dampen foreign antigen specific immunity. This report extends our previous findings by demonstrating that immunity to certain herpes simplex virus (HSV) vaccines is significantly elevated and more effective if T(reg) cell response is curtailed during either primary or recall immunization. The data presented here show that removal of CD25(+) T(reg) cells prior to SSIEFARL-CpG or gB-DNA immunization significantly enhanced the resultant CD8(+) T-cell response to the immunodominant SSIEFARL peptide. The enhanced CD8(+) T-cell reactivity in T(reg) cell-depleted animals was between two- and threefold and evident in both acute and memory stages. Interestingly, removal of CD25(+) T(reg) cells during the memory recall response to plasmid immunization resulted in a twofold increase in CD8(+) T-cell memory pool. Moreover, in the challenge experiments, memory CD8(+) T cells generated with plasmid DNA in the absence of T(reg) cells cleared the virus more effectively compared with control groups. We conclude that CD25(+) T(reg) cells quantitatively as well as qualitatively affect the memory CD8(+) T-cell response generated by gB-DNA vaccination against HSV. However, it remains to be seen if all types of vaccines against HSV are similarly affected by CD25(+) T(reg) cells and if it is possible to devise means of limiting T(reg) cell activity to enhance vaccine efficacy.     

Two Overlapping Subdominant Epitopes Identified by DNA Immunization Induce Protective CD8+ T-Cell Populations with Differing Cytolytic Activities

Subdominant CD8(+) T-cell responses contribute to control of several viral infections and to vaccine-induced immunity. Here, using the lymphocytic choriomeningitis virus model, we demonstrate that subdominant epitopes can be more reliably identified by DNA immunization than by other methods, permitting the identification, in the virus nucleoprotein, of two overlapping subdominant epitopes: one presented by L(d) and the other presented by K(d). This subdominant sequence confers immunity as effective as that induced by the dominant epitope, against which >90% of the antiviral CD8(+) T cells are normally directed. We compare the kinetics of the dominant and subdominant responses after vaccination with those following subsequent viral infection. The dominant CD8(+) response expands more rapidly than the subdominant responses, but after virus infection is cleared, mice which had been immunized with the "dominant" vaccine have a pool of memory T cells focused almost entirely upon the dominant epitope. In contrast, after virus infection, mice which had been immunized with the "subdominant" vaccine retain both dominant and subdominant memory cells. During the acute phase of the immune response, the acquisition of cytokine responsiveness by subdominant CD8(+) T cells precedes their development of lytic activity. Furthermore, in both dominant and subdominant populations, lytic activity declines more rapidly than cytokine responsiveness. Thus, the lysis(low)-cytokine(competent) phenotype associated with most memory CD8(+) T cells appears to develop soon after antigen clearance. Finally, lytic activity differs among CD8(+) T-cell populations with different epitope specificities, suggesting that vaccines can be designed to selectively induce CD8(+) T cells with distinct functional attributes.     

CD4-directed peptide vaccination augments an antitumor response, but efficacy is limited by the number of CD8+ T cell precursors

Peptide vaccination is an immunotherapeutic strategy being pursued as a method of enhancing Ag-specific antitumor responses. To date, most studies have focused on the use of MHC class I-restricted peptides, and have not shown a correlation between Ag-specific CD8(+) T cell expansion and the generation of protective immune responses. We investigated the effects of CD4-directed peptide vaccination on the ability of CD8(+) T cells to mount protective antitumor responses in the DUC18/CMS5 tumor model system. To accomplish this, we extended the amino acid sequence of the known MHC class I-restricted DUC18 rejection epitope from CMS5 to allow binding to MHC class II molecules. Immunization with this peptide (tumor-derived extracellular signal-regulated kinase-II (tERK-II)) induced Ag-specific CD4(+) T cell effector function, but did not directly prime CD8(+) T cells. Approximately 31% of BALB/c mice immunized with tERK-II were protected from subsequent tumor challenge in a CD40-dependent manner. Priming of endogenous CD8(+) T cells in immunized mice was detected only after CMS5 challenge. Heightened CD4(+) Th cell function in response to tERK II vaccination allowed a 12-fold reduction in the number of adoptively transferred CD8(+) DUC18 T cells needed to protect recipients against tumor challenge as compared with previous studies using unimmunized mice. Furthermore, tERK-II immunization led to a more rapid and transient expansion of transferred DUC18 T cells than was seen in unimmunized mice. These findings illustrate that CD4-directed peptide vaccination augments antitumor immunity, but that the number of tumor-specific precursor CD8(+) T cells will ultimately dictate the success of immunotherapy.     

In vivo evidence that peptide vaccination can induce HLA-DR-restricted CD4+ T cells reactive to a class I tumor peptide

Vaccination with class I tumor peptides has been performed to induce tumor-reactive CD8(+) T cells in vivo. However, the kinds of immune responses that vaccination might elicit in patients are not fully understood. In this study we tried to elucidate the mechanisms by which vaccination of class I binding tumor peptides into an HLA-A2(+) lung cancer patient elicited dramatic amounts of IgG1 and IgG2 specific to a nonamer peptide, ubiquitin-conjugated enzyme variant Kua (UBE2V)(43-51). The UBE2V(43-51) peptide contains cysteine at the sixth position. HLA-DR-restricted and UBE2V(43-51) peptide-recognizing CD4(+) T cells were induced from postvaccination, but not from prevaccination, PBMCs of the cancer patient. In addition, a CD4(+) T cell line (UB-2) and its clone (UB-2.3), both of which recognize the UBE2V(43-51) peptide in the context of HLA-DRB1*0403 molecules, were successfully established from postvaccination PBMCs. The peptide vaccination increased the frequency of peptide-specific T cells, especially CD4(+) T cells. In contrast, mass spectrometric analysis revealed that the vaccinated UBE2V(43-51) peptide contained both monomeric and dimeric forms. Both forms, fractionated by reverse phase HPLC, were recognized by UB-2 and UB-2.3 cells. Recognition by these CD4(+) T cells was observed despite the addition of a reduction reagent or the fixation of APC. Overall, these results indicate that vaccination with class I tumor peptides can induce HLA-DR-restricted CD4(+) T cells in vivo and elicit humoral immune responses, and that a cysteine-containing peptide can be recognized by CD4(+) T cells not only as a monomer, but also as a dimer.