Enhancement of gp120-specific immune responses by genetic vaccination with the human immunodeficiency virus type 1 envelope gene fused to the gene coding for soluble CTLA4

DNA vaccines exploit the inherent abilities of professional antigen-presenting cells to prime the immune system and to elicit immunity against diverse pathogens. In this study, we explored the possibility of augmenting human immunodeficiency virus type 1 gp120-specific immune responses by a DNA vaccine coding for a fusion protein, CTLA4:gp120, in mice. In vitro binding studies revealed that secreted CTLA4:gp120 protein induced a mean florescence intensity shift, when incubated with Raji B cells, indicating its binding to B7 proteins on Raji B cells. Importantly, we instituted three different vaccination regimens to test the efficacy of DNA vaccines encoding gp120 and CTLA4:gp120 in the induction of both cellular (CD8(+)) and antibody responses. Each of the vaccination regimens incorporated a single intramuscular (i.m.) injection of the DNA vaccines to prime the immune system, followed by two booster injections. The i.m.-i.m.-i.m. regimen induced only modest levels of gp120-specific CD8(+) T cells, but the antibody response by CTLA4:gp120 DNA was nearly 16-fold higher than that induced by gp120 DNA. In contrast, using the i.m.-subcutaneous (s.c.)-i.m. regimen, it was found that gp120 and CTLA4:gp120 DNAs were capable of inducing significant levels of gp120-specific CD8(+) T cells (3.5 and 11%), with antibody titers showing a modest twofold increase for CTLA4:gp120 DNA. In the i.m.-gene gun (g.g.)-g.g. regimen, the mice immunized with gp120 and CTLA4:gp120 harbored gp120-specific CD8(+) T cells at frequencies of 0.9 and 2.9%, with the latter showing an eightfold increase in antibody titers. Thus, covalent antigen modification and the routes of genetic vaccination have the potential to modulate antigen-specific immune responses in mice.     

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.     

Effective genetic vaccination with a widely shared endogenous retroviral tumor antigen requires CD40 stimulation during tumor rejection phase

Endogenous retrovirus (ERV) products are recognized by T lymphocytes in mice and humans. As these Ags are preferentially expressed by neoplastic tissues, they might represent an ideal target for active immunization by genetic vaccination. However, i.m. inoculation of plasmid DNA encoding mouse gp70 or p15E, two products of the env gene of an endogenous murine leukemia virus, elicited a weak Ag-specific T lymphocyte response and resulted in partial protection from challenge with mouse tumors possessing these Ags. Depletion experiments showed that CD8(+), but not CD4(+), T lymphocytes were crucial for the antitumor activity of the vaccines. Systemic administration of agonistic anti-CD40 mAb increased the therapeutic potential of genetic vaccination, but only when given during the tumor rejection phase and not at the time of immunization. This effect correlated with a dramatic increase in the number of ERV-specific CD8(+) T lymphocytes. Adjuvant activity of CD40 agonists thus seems to be relevant to enhance the CD8(+) T cell-dependent response in tumor-bearing hosts, suggesting that sustaining tumor-specific T lymphocyte survival in subjects undergoing vaccination might be a key event in the successful vaccination with weak tumor Ags.     

Potent CD4+ T cell responses elicited by a bicistronic HIV-1 DNA vaccine expressing gp120 and GM-CSF.

Virus-specific CD4(+) T cell responses have been shown to play a critical role in controlling HIV-1 replication. Candidate HIV-1 vaccines should therefore elicit potent CD4(+) as well as CD8(+) T cell responses. In this report we investigate the ability of plasmid GM-CSF to augment CD4(+) T cell responses elicited by an HIV-1 gp120 DNA vaccine in mice. Coadministration of a plasmid expressing GM-CSF with the gp120 DNA vaccine led to only a marginal increase in gp120-specific splenocyte CD4(+) T cell responses. However, immunization with a bicistronic plasmid that coexpressed gp120 and GM-CSF under control of a single promoter led to a dramatic augmentation of vaccine-elicited CD4(+) T cell responses, as measured by both cellular proliferation and ELISPOT assays. This augmentation of CD4(+) T cell responses was selective, since vaccine-elicited Ab and CD8(+) T cell responses were not significantly changed by the addition of GM-CSF. A 100-fold lower dose of the gp120/GM-CSF bicistronic DNA vaccine was required to elicit detectable gp120-specific splenocyte proliferative responses compared with the monocistronic gp120 DNA vaccine. Consistent with these findings, i.m. injection of the gp120/GM-CSF bicistronic DNA vaccine evoked a more extensive cellular infiltrate at the site of inoculation than the monocistronic gp120 DNA vaccine. These results demonstrate that bicistronic DNA vaccines containing GM-CSF elicit remarkably potent CD4(+) T cell responses and suggest that optimal Th cell priming requires the precise temporal and spatial codelivery of Ag and GM-CSF.     

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.     

Coimmunization with an optimized IL-15 plasmid results in enhanced function and longevity of CD8 T cells that are partially independent of CD4 T cell help

DNA vaccines are a promising technology for the induction of Ag-specific immune responses, and much recent attention has gone into improving their immune potency. In this study we test the feasibility of delivering a plasmid encoding IL-15 as a DNA vaccine adjuvant for the induction of improved Ag-specific CD8(+) T cellular immune responses. Because native IL-15 is poorly expressed, we used PCR-based strategies to develop an optimized construct that expresses 80-fold higher than the native IL-15 construct. Using a DNA vaccination model, we determined that immunization with optimized IL-15 in combination with HIV-1gag DNA constructs resulted in a significant enhancement of Ag-specific CD8(+) T cell proliferation and IFN-gamma secretion, and strong induction of long-lived CD8(+) T cell responses. In an influenza DNA vaccine model, coimmunization with plasmid expressing influenza A PR8/34 hemagglutinin with the optimized IL-15 plasmid generated improved long term CD8(+) T cellular immunity and protected the mice against a lethal mucosal challenge with influenza virus. Because we observed that IL-15 appeared to mostly adjuvant CD8(+) T cell function, we show that in the partial, but not total, absence of CD4(+) T cell help, plasmid-delivered IL-15 could restore CD8 secondary immune responses to an antigenic DNA plasmid, supporting the idea that the effects of IL-15 on CD8(+) T cell expansion require the presence of low levels of CD4 T cells. These data suggest a role for enhanced plasmid IL-15 as a candidate adjuvant for vaccine or immunotherapeutic studies.     

Enhancing DNA vaccine potency by combining a strategy to prolong dendritic cell life with intracellular targeting strategies

We have recently shown that intradermal coadministration of DNA encoding Ag with DNA encoding inhibitors of apoptosis, including Bcl-x(L), prolongs dendritic cell (DC) life and thereby enhances the potency of DNA vaccines in vivo. We have also demonstrated that DNA vaccines targeting Ag to subcellular compartments, using proteins such as Mycobacterium tuberculosis heat shock protein 70, calreticulin, or the sorting signal of the lysosome-associated membrane protein type 1 (LAMP-1), enhanced DNA vaccine potency. In this study, we reasoned that the combination of a strategy to prolong DC life with intracellular targeting strategies might produce a more effective DNA vaccine against human papillomavirus E7. We showed that coadministration of DNA encoding Bcl-x(L) with DNA encoding E7/heat shock protein 70, calreticulin/E7, or Sig/E7/LAMP-1 resulted in further enhancement of the E7-specific CD8(+) T cell response for all three constructs. Of these strategies, mice vaccinated with Sig/E7/LAMP-1 DNA mixed with Bcl-x(L) DNA showed the greatest increase in E7-specific CD8(+) T cells ( approximately 13-fold increase). This combination of strategies resulted in increased CD8(+) T cell functional avidity, an increased E7-specific CD4(+) Th1 cell response, enhanced tumor treatment ability, and stronger long-term tumor protection when compared with mice vaccinated without Bcl-x(L) DNA. Therefore, DNA vaccines that combine strategies to enhance intracellular Ag processing and prolong DC life have potential clinical implications for control of viral infection and neoplasia.     

Vaccination with a Shigella DNA vaccine vector induces antigen-specific CD8(+) T cells and antiviral protective immunity

A prototype Shigella human immunodeficiency virus type 1 (HIV-1) gp120 DNA vaccine vector was constructed and evaluated for immunogenicity in a murine model. For comparative purposes, mice were also vaccinated with a vaccinia virus-env (vaccinia-env) vector or the gp120 DNA vaccine alone. Enumeration of the CD8(+)-T-cell responses to gp120 after vaccination using a gamma interferon enzyme-linked spot assay revealed that a single intranasal dose of the Shigella HIV-1 gp120 DNA vaccine vector elicited a CD8(+) T-cell response to gp120, the magnitude of which was comparable to the sizes of the analogous responses to gp120 that developed in mice vaccinated intraperitoneally with the vaccinia-env vector or intramuscularly with the gp120 DNA vaccine. In addition, a single dose of the Shigella gp120 DNA vaccine vector afforded significant protection against a vaccinia-env challenge. Moreover, the number of vaccinia-env PFU recovered in mice vaccinated intranasally with the Shigella vector was about fivefold less than the number recovered from mice vaccinated intramuscularly with the gp120 DNA vaccine. Since the Shigella vector did not express detectable levels of gp120, this report confirms that Shigella vectors are capable of delivering passenger DNA vaccines to host cells and inducing robust CD8(+) T-cell responses to antigens expressed by the DNA vaccines. Furthermore, to our knowledge, this is the first documentation of antiviral protective immunity following vaccination with a live Shigella DNA vaccine vector.     

Subsets of memory cytotoxic T lymphocytes elicited by vaccination influence the efficiency of secondary expansion in vivo

Vaccine-elicited cytotoxic T lymphocytes (CTL) should be long-lived memory cells that can rapidly expand in number following re-exposure to antigen. The present studies were initiated to analyze the ability of plasmid interleukin-12 (IL-12) to augment CTL responses in mice when delivered during the peak phase of an immune response elicited by a plasmid human immunodeficiency virus type 1 gp120 DNA vaccine. Delivery of plasmid IL-12 on day 10 postimmunization resulted in a robust expansion of gp120-specific CD8+ T cells, as measured by tetramer, gamma interferon ELISPOT, and functional-killing assays. Interestingly, this delayed administration of plasmid IL-12 had no significant effect on antigen-specific CD4(+)-T-cell and antibody responses. Phenotypic analyses suggested that administration of plasmid IL-12 near the time of the peak CTL response activated and expanded antigen-specific effector cells, preventing their loss through apoptosis. However, this IL-12-augmented population of gp120-specific CD8+ T cells did not efficiently expand following gp120 boost immunization, suggesting that these effector cells would be of little utility in expanding to contain a viral infection. Analyses of the phenotypic profile and anatomic distribution of the plasmid IL-12-augmented CTL population indicated that these lymphocytes were primarily effector memory rather than central memory T cells. These observations suggest that CTL-based vaccines should elicit central memory rather than effector memory T cells and illustrate the importance of monitoring the phenotype and functionality of vaccine-induced, antigen-specific CTL.     

DNA-encoded fetal liver tyrosine kinase 3 ligand and granulocyte macrophage-colony-stimulating factor increase dendritic cell recruitment to the inoculation site and enhance antigen-specific CD4+ T cell responses induced by DNA vaccination of outbred animals

DNA-based immunization is a contemporary strategy for developing vaccines to prevent infectious diseases in animals and humans. Translating the efficacy of DNA immunization demonstrated in murine models to the animal species that represent the actual populations to be protected remains a significant challenge. We tested two hypotheses directed at enhancing DNA vaccine efficacy in outbred animals. The first hypothesis, that DNA-encoding fetal liver tyrosine kinase 3 ligand (Flt3L) and GM-CSF increases dendritic cell (DC) recruitment to the immunization site, was tested by intradermal inoculation of calves with plasmid DNA encoding Flt3L and GM-CSF followed by quantitation of CD1(+) DC. Peak DC recruitment was detected at 10-15 days postinoculation and was significantly greater (p < 0.05) in calves in the treatment group as compared with control calves inoculated identically, but without Flt3L and GM-CSF. The second hypothesis, that DNA encoding Flt3L and GM-CSF enhances immunity to a DNA vector-expressed Ag, was tested by analyzing the CD4(+) T lymphocyte response to Anaplasma marginale major surface protein 1a (MSP1a). Calves immunized with DNA-expressing MSP1a developed strong CD4(+) T cell responses against A. marginale, MSP1a, and specific MHC class II DR-restricted MSP1a epitopes. Administration of DNA-encoding Flt3L and GM-CSF before MSP1a DNA vaccination significantly increased the population of Ag-specific effector/memory cells in PBMC and significantly enhanced MSP1a-specific CD4(+) T cell proliferation and IFN-gamma secretion as compared with MHC class II DR-matched calves vaccinated identically but without Flt3L and GM-CSF. These results support use of these growth factors in DNA vaccination and specifically indicate their applicability for vaccine testing in outbred animals.     

Safety and immunogenicity of ALVAC vCP1452 and recombinant gp160 in newly human immunodeficiency virus type 1-infected patients treated with prolonged highly active antiretroviral therapy

In order to boost immune responses in persons in whom highly active antiretroviral therapy (HAART) was initiated within 120 days of the onset of symptoms of newly acquired human immunodeficiency virus type 1 (HIV-1) infection, we administered vaccines containing a canarypox virus vector, vCP1452, with HIV-1 genes encoding multiple HIV-1 proteins, and recombinant gp160. Fifteen HIV-1-infected subjects who achieved sustained suppression of plasma viremia for at least 2 years were enrolled. While continuing antiretroviral therapy, each subject received at least four intramuscular injections of the vaccines on days 0, 30, 90, and 180. Adverse events were mild, with the most common being transient tenderness at the vCP1452 injection site. Of the 14 patients who completed vaccination, 13 had significant increases in anti-gp120 or anti-p24 antibody titers, and 9 had transient augmentation of their T-cell proliferation responses to gp160 and/or p24. HIV-1-specific CD8(+) T cells were quantified using an intracellular gamma interferon staining assay. Among 11 patients who had increased CD8(+) T-cell responses, seven had responses to more than one HIV-1 antigen. In summary, vaccination with vCP1452 and recombinant gp160 appears safe and immunogenic in newly HIV-1-infected patients on HAART.     

Induction of potent CD8+ T-cell responses by novel biodegradable nanoparticles carrying human immunodeficiency virus type 1 gp120

The mainstream of recent anti-AIDS vaccines is a prime/boost approach with multiple doses of the target DNA of human immunodeficiency virus type 1 (HIV-1) and recombinant viral vectors. In this study, we have attempted to construct an efficient protein-based vaccine using biodegradable poly(gamma-glutamic acid) (gamma-PGA) nanoparticles (NPs), which are capable of inducing potent cellular immunity. A significant expansion of CD8+ T cells specific to the major histocompatibility complex class I-restricted gp120 epitope was observed in mice intranasally immunized once with gp120-carrying NPs but not with gp120 alone or gp120 together with the B-subunit of cholera toxin. Both the gp120-encapsulating and -immobilizing forms of NPs could induce antigen-specific spleen CD8+ T cells having a functional profile of cytotoxic T lymphocytes. Long-lived memory CD8+ T cells could also be elicited. Although a substantial decay in the effector memory T cells was observed over time in the immunized mice, the central memory T cells remained relatively constant from day 30 to day 238 after immunization. Furthermore, the memory CD8+ T cells rapidly expanded with boosting with the same immunogen. In addition, gamma-PGA NPs were found to be a much stronger inducer of antigen-specific CD8+ T-cell responses than nonbiodegradable polystyrene NPs. Thus, gamma-PGA NPs carrying various HIV-1 antigens may have great potential as a novel priming and/or boosting tool in current vaccination regimens for the induction of cellular immune responses.     

A DNA vaccine co-expressing antigen and an anti-apoptotic molecule further enhances the antigen-specific CD8+ T-cell immune response

We have shown that DNA encoding the anti-apoptotic protein Bcl-xL enhances E7-specific CD8+ T-cell responses and DNA encoding pro-apoptotic protein caspase-3 suppresses E7-specific CD8+ T-cell responses when co-administered intradermally via gene gun with DNA encoding human papillomavirus type 16 (HPV-16) E7 linked to the sorting signal of the lysosome-associated membrane protein type 1 (LAMP-1). E7 and LAMP-1 are linked to form the chimeric Sig/E7/LAMP-1 (SEL). Because co-administration does not ensure delivery of both constructs to a single cell, we used pVITRO, a mammalian expression vector with double promoters, to ensure expression of both molecules in the same cell. We vaccinated C57BL/6 mice with pVITRO-SEL-Bcl-xL, pVITRO-SEL-mtBcl-xL, pVITRO-SEL, or pVITRO-SEL-caspase-3 intradermally via gene gun and intramuscularly via injection. We demonstrated that vaccination with pVITRO achieved similar results to a co-administration strategy: that Bcl-xL enhanced the E7-specific CTL response and caspase-3 suppressed the E7-specific CTL response. In addition, we found intradermal vaccination elicited significantly higher numbers of E7-specific CD8+ T cells compared to intramuscular vaccination. Thus, intradermal vaccination with a pVITRO vector combining an anti-apoptotic strategy (Bcl-xL) and an intracellular targeting strategy (SEL) further enhances the E7-specific CD8+ T-cell response and guarantees co-expression of both encoded molecules in transfected cells.T.W.K. and C.-F.H. contributed equally to this work.     

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.     

CD8(+) T-cell homeostasis after infection: setting the 'curve'

Antigen (Ag)-specific CD8(+) T-cell responses exhibit remarkably similar kinetics after different types of infection. Starting from levels that are virtually undetectable in vivo, pathogen-specific na?ve CD8(+) T cells are precisely regulated to go through rapid expansion and contraction (death) phases, achieving memory levels of Ag-specific CD8(+) T cells that are maintained for the life of the host. However, the exact mechanisms used to achieve appropriate and reproducible CD8(+) T-cell homeostasis in response to diverse pathogens remain to be determined. The possibility that early events after infection regulate major features of Ag-specific CD8(+) T-cell homeostasis will be discussed here.     

Critical components of a DNA fusion vaccine able to induce protective cytotoxic T cells against a single epitope of a tumor antigen

DNA vaccines can activate immunity against tumor Ags expressed as MHC class I-associated peptides. However, priming of CD8(+) CTL against weak tumor Ags may require adjuvant molecules. We have used a pathogen-derived sequence from tetanus toxin (fragment C (FrC)) fused to tumor Ag sequences to promote Ab and CD4(+) T cell responses. For induction of CD8(+) T cell responses, the FrC sequence has been engineered to remove potentially competitive MHC class I-binding epitopes and to improve presentation of tumor epitopes. The colon carcinoma CT26 expresses an endogenous retroviral gene product, gp70, containing a known H2-L(d)-restricted epitope (AH1). A DNA vaccine encoding gp70 alone was a poor inducer of CTL, and performance was not significantly improved by fusion of full-length FrC. However, use of a minimized domain of FrC, with the AH1 sequence fused to the 3' position, led to rapid induction of high levels of CTL. IFN-gamma-producing epitope-specific CTL were detectable ex vivo and these killed CT26 targets in vitro. The single epitope vaccine was more effective than GM-CSF-transfected CT26 tumor cells in inducing an AH1-specific CTL response and equally effective in providing protection against tumor challenge. Levels of AH1-specific CTL in vivo were increased following injection of tumor cells, and CTL expanded in vitro were able to kill CT26 cells in tumor bearers. Pre-existing immunity to tetanus toxoid had no effect on the induction of AH1-specific CTL. These data demonstrate the power of epitope-specific CTL against tumor cells and illustrate a strategy for priming immunity via a dual component DNA vaccine.     

Recognition of HIV glycoprotein gp120 by T cells. Role of monocyte CD4 in the presentation of gp120

The HIV envelope glycoprotein gp120 binds with high affinity to CD4 and is responsible for the tropism of HIV for CD4+ T cells and monocytes. Efforts to develop HIV vaccines have focused on gp120 and, therefore, a detailed molecular understanding of human immune responses to gp120 is essential. In this report, we have used human T cell clones specific for gp120 to examine the processing and presentation of gp120 to T cells. In particular, we examined the role of the CD4 that is expressed at low levels on the surfaces of human monocytes in the presentation of gp120 by monocytes. The presentation of gp120 to gp120-specific human T cell clones was blocked by pretreatment of monocytes with anti-CD4 mAb. Blocking of monocyte CD4 with anti-CD4 did not inhibit presentation of other Ag or of synthetic peptides representing epitopes within gp120 recognized by gp120-specific T cell clones. These results indicated that the anti-CD4-mediated inhibition occurred at the level of the monocyte, was specific for the gp120 response, and was operative at the initial Ag uptake phase of the Ag-processing pathway. Definitive confirmation that monocyte CD4 functions in the initial uptake step of the gp120-processing pathway was obtained by using soluble CD4 to block the interaction of gp120 with monocyte CD4. These results demonstrate that gp120 expressed by human monocytes plays an important role in the initial uptake of gp120 by monocytes and that gp120 taken up via CD4 is subsequently processed to allow for exposure of epitopes recognized by gp120-specific human T cells. At limiting gp120 concentrations, uptake via CD4 is essential for the presentation of gp120.     

Enhancement of DNA vaccine potency through coadministration of CIITA DNA with DNA vaccines via gene gun

Administration of DNA vaccines via gene gun has emerged as an important form of Ag-specific immunotherapy. The MHC CIITA is a master regulator of MHC class II expression and also induces expression of class I molecules. We reasoned that the gene gun administration of CIITA DNA with DNA vaccines employing different strategies to improve MHC I and II processing could enhance DNA vaccine potency. We observed that DC-1 cells transfected with CIITA DNA lead to higher expression of MHC I and II molecules, leading to enhanced Ag presentation through the MHC I/II pathways. Furthermore, our data suggested that coadministration of DNA-encoding calreticulin (CRT) linked to human papillomavirus (HPV) 16 E6 Ag (CRT/E6) with CIITA DNA leads to enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. In addition, coadministration of the combination of CRT/E6 DNA with CIITA DNA and DNA encoding the invariant chain (Ii) linked to the pan HLA-DR-reactive epitope (Ii-PADRE) further enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. Treatment with the combination vaccine was also shown to enhance the antitumor effects and to prolong survival in TC-1 tumor-bearing mice. Vaccination with the combination vaccine also led to enhanced E6-specific CD8(+) memory T cells and to long-term protection against TC-1 tumors and prolonged survival in vaccinated mice. Thus, our findings suggest that the combination of CIITA DNA with CRT/E6 and Ii-PADRE DNA vaccines represents a potentially effective means to combat tumors in the clinical setting.     

Rapid clonal expansion and prolonged maintenance of memory CD8+ T cells of the effector (CD44highCD62Llow) and central (CD44highCD62Lhigh) phenotype by an archaeosome adjuvant independent of TLR2

Vaccines capable of eliciting long-term T cell immunity are required for combating many diseases. Live vectors can be unsafe whereas subunit vaccines often lack potency. We previously reported induction of CD8(+) T cells to Ag entrapped in archaeal glycerolipid vesicles (archaeosomes). In this study, we evaluated the priming, phenotype, and functionality of the CD8(+) T cells induced after immunization of mice with OVA-Methanobrevibacter smithii archaeosomes (MS-OVA). A single injection of MS-OVA evoked a profound primary response but the numbers of H-2K(b)OVA(257-264)-specific CD8(+) T cells declined by 14-21 days, and <1% of primarily central phenotype (CD44(high)CD62L(high)) cells persisted. A booster injection of MS-OVA at 3-11 wk promoted massive clonal expansion and a peak effector response of approximately 20% splenic/blood OVA(257-264)-specific CD8(+) T cells. Furthermore, contraction was protracted and the memory pool (IL-7Ralpha(high)) of approximately 5% included effector (CD44(high)CD62L(low)) and central (CD44(high)CD62L(high)) phenotype cells. Recall response was observed even at >300 days. CFSE-labeled naive OT-1 (OVA(257-264) TCR transgenic) cells transferred into MS-OVA-immunized recipients cycled profoundly (>90%) within the first week of immunization indicating potent Ag presentation. Moreover, approximately 25% cycling of Ag-specific cells was seen for >50 days, suggesting an Ag depot. In vivo, CD8(+) T cells evoked by MS-OVA killed >80% of specific targets, even at day 180. MS-OVA induced responses similar in magnitude to Listeria monocytogenes-OVA, a potent live vector. Furthermore, protective CD8(+) T cells were induced in TLR2-deficient mice, suggesting nonengagement of TLR2 by archaeal lipids. Thus, an archaeosome adjuvant vaccine represents an alternative to live vectors for inducing CD8(+) T cell memory.