Adjuvantive effects of anti-4-1BB agonist Ab and 4-1BBL DNA for a HIV-1 Gag DNA vaccine: Different effects on cellular and humoral immunity

Plasmid DNA immunizations induce low levels but a broad spectrum of cellular and humoral immune responses. Here, we investigate the potential of co-stimulation through 4-1BB as an adjuvant for a HIV-1 DNA vaccine in mice. We designed plasmid DNAs expressing either the membrane bound or soluble form of 4-1BBL, and compared with the agonistic anti-4-1BB Ab for their ability to adjuvant the Gag DNA vaccine. Both, anti-4-1BB agonistic Ab as well as 4-1BBL DNA enhanced the Gag-specific cellular immune responses. However, in complete contrast to the agonistic Ab that suppressed humoral immunity to Gag, 4-1BBL DNA adjuvanted vaccines enhanced Gag-specific IgG responses. Importantly, the expression of Gag and 4-1BBL from the same plasmid was critical for the adjuvant activity. Collectively, our data suggest that for a HIV-1 vaccine where both antigen-specific cellular and humoral immunity are desirable, 4-1BBL expressed by a DNA vaccine is a superior adjuvant than anti-4-1BB agonistic Ab.     

A prime–boost immunisation regimen using recombinant BCG and Pr55 virus-like particle vaccines based on HIV type 1 subtype C successfully elicits Gag-specific responses in baboons

Mycobacterium bovis BCG is considered an attractive live bacterial vaccine vector. In this study, we investigated the immune response of baboons to a primary vaccination with recombinant BCG (rBCG) constructs expressing the gag gene from a South African HIV-1 subtype C isolate, and a boost with HIV-1 subtype C Pr55^gag virus-like particles (Gag VLPs). Using an interferon enzyme-linked immunospot assay, we show that although these rBCG induced only a weak or an undetectable HIV-1 Gag-specific response on their own, they efficiently primed for a Gag VLP boost, which strengthened and broadened the immune responses. These responses were predominantly CD8+ T cell-mediated and recognised similar epitopes as those targeted by humans with early HIV-1 subtype C infection. In addition, a Gag-specific humoral response was elicited. These data support the development of HIV-1 vaccines based on rBCG and Pr55^gag VLPs.     

4-1BBL coexpression enhances HIV-specific CD8 T cell memory in a poxvirus prime-boost vaccine

We have constructed a recombinant fowlpox virus expressing HIV antigens and the costimulatory molecule 4-1BBL. When included in the boost, but not the prime of a poxvirus prime-boost strategy, 4-1BBL significantly enhanced the anti-HIV T cell response generated to this vaccination in BALB/c mice, as detected by ex vivo IFNgamma ELISPOT responses, intracellular cytokine staining to HIV Gag antigens, and enumeration of Gag-reactive CD8 T cells. 4-1BBL however, is not capable of modulating the CD4 T cell response, nor the antibody response to this vaccination strategy. Enhancement of the T cell response by 4-1BBL continues into the memory phase, as detected 2 months post vaccination. This data is the first to show modulation of the immune response to a viral vaccine by coexpression of 4-1BBL and supports this strategy as an exciting approach for enhancement of T cell memory in prime-boost vaccines.     

Improving the Th1 cellular efficacy of the lead Yersinia pestis rF1-V subunit vaccine using SA-4-1BBL as a novel adjuvant

The lead candidate plague subunit vaccine is the recombinant fusion protein rF1-V adjuvanted with alum. While alum generates Th₂ regulated robust humoral responses, immune protection against Yersinia pestis has been shown to also involve Th₁ driven cellular responses. Therefore, the rF1-V-based subunit vaccine may benefit from an adjuvant system that generates a mixed Th₁ and humoral immune response. We herein assessed the efficacy of a novel SA-4-1BBL costimulatory molecule as a Th₁ adjuvant to improve cellular responses generated by the rF1-V vaccine. SA-4-1BBL as a single adjuvant had better efficacy than alum in generating CD4⁺ and CD8⁺ T cells producing TNFα and IFNγ, signature cytokines for Th₁ responses. The combination of SA-4-1BBL with alum further increased this Th₁ response as compared with the individual adjuvants. Analysis of the humoral response revealed that SA-4-1BBL as a single adjuvant did not generate a significant Ab response against rF1-V, and SA-4-1BBL in combination with alum did not improve Ab titers. However, the combined adjuvants significantly increased the ratio of Th₁ regulated IgG_2c in C57BL/6 mice to the Th₂ regulated IgG₁. Finally, a single vaccination with rF1-V adjuvanted with SA-4-1BBL + alum had better protective efficacy than vaccines containing individual adjuvants. Taken together, these results demonstrate that SA-4-1BBL improves the protective efficacy of the alum adjuvanted lead rF1-V subunit vaccine by generating a more balanced Th₁ cellular and humoral immune response. As such, this adjuvant platform may prove efficacious not only for the rF1-V vaccine but also against other infections that require both cellular and humoral immune responses for protection.     

SIV antigen-specific effects on immune responses induced by vaccination with DNA electroporation and plasmid IL-12

Molecular adjuvants are important for augmenting or modulating immune responses induced by DNA vaccination. Promising results have been obtained using IL-12 expression plasmids in a variety of disease models including the SIV model of HIV infection. We used a mouse model to evaluate plasmid IL-12 (pIL-12) in a DNA prime, recombinant adenovirus serotype 5 (rAd5) boost regimen specifically to evaluate the effect of IL-12 expression on cellular and humoral immunity induced against both SIVmac239 Gag and Env antigens. Priming with electroporated (EP) DNA + pIL-12 resulted in a 2–4-fold enhanced frequency of Gag-specific CD4 T cells which was maintained through the end of the study irrespective of the pIL-12 dose, while memory Env-specific CD4 + T cells were maintained only at the low dose of pIL-12. There was little positive effect of pIL-12 on the humoral response to Env, and in fact, high dose pIL-12 dramatically reduced SIV Env-specific IgG. Additionally, both doses of pIL-12 diminished the frequency of CD8 T-cells after DNA prime, although a rAd5 boost recovered CD8 responses regardless of the pIL-12 dose. In this prime-boost regimen, we have shown that a high dose pIL-12 can systemically reduce Env-specific humoral responses and CD4T cell frequency, but not Gag-specific CD4+ T cells. These data indicate that it is important to independently characterize individual SIV or HIV antigen immunogenicity in multi-antigenic vaccines as a function of adjuvant dose.     

DNA prime/MVTT boost regimen with HIV-1 mosaic Gag enhances the potency of antigen-specific immune responses

HIV-1 diversity and latent reservoir are the major challenges for the development of an effective AIDS vaccine. It is well indicated that Gag-specific CD8⁺ T cells serve as the dominant host immune surveillance for HIV-1 control, but it still remains a challenge for vaccine design to induce broader and stronger cytotoxic T cell immunity against the virus. Genetic variation of the HIV-1 gag gene across different clades is one of the reasons for the reduction of antigenic epitope coverage. Here, we report an immunization strategy with heterologous vaccines expressing a mosaic Gag antigen aimed to increase antigenic breadth against a wider spectrum of HIV-1 strains. Priming using a DNA vaccine via in vivo electroporation, followed by boosting with a live replication-competent modified vaccinia TianTan (MVTT) vectored vaccine, elicited greater and broader protective Gag-specific immune responses in mice. Compared to DNA or MVTT homologous immunization, the heterologous DNA/MVTT vaccination resulted in higher frequencies of broadly reactive, Gag-specific, polyfunctional, long-lived cytotoxic CD8⁺ T cells, as well as increased anti-Gag antibody titer. Importantly, the DNA/MVTT heterologous vaccination induced protection against EcoHIV and mesothelioma AB1-Gag challenges. In summary, the stronger protective Gag-specific immunity induced by the heterologous regimen using two safe vectors shows promise for further development to enhance anti-HIV-1 immunity. Our study has important implications for immunogen design and the development of an effective HIV-1 heterologous vaccination strategy.     

The improved antibody response against HIV-1 after a vaccination based on intrastructural help is complemented by functional CD8+ T cell responses

Despite more than three decades of intense research, a prophylactic HIV-1 vaccine remains elusive. Four vaccine modalities have been evaluated in clinical efficacy studies, but only one demonstrated at least modest efficacy, which correlated with polyfunctional antibody responses to the HIV surface protein Env. To be most effective, a HIV-1 vaccine probably has to induce both, functional antibody and CD8⁺ T cell responses. We therefore analyzed DNA/DNA and DNA/virus-like particle (VLP) regimens for their ability to induce humoral and cellular immune responses. Here, DNA vaccination of mice induced strong CD8⁺ responses against Env and Gag. However, the humoral response to Env was dominated by IgG1, a subclass known for its low functionality. In contrast, priming only with the Gag-encoding plasmid followed by a boost with VLPs consisting of Gag and Env improved the quality of the anti-Env antibody response via intrastructural help (ISH) provided by Gag-specific T cells to Env-specific B cells. Furthermore, the Gag-specific CD8⁺ T cells induced by the DNA prime immunization could still protect from a lethal infection with recombinant vaccinia virus encoding HIV Gag. Therefore, this immunization regimen represents a promising approach to combine functional antibody responses toward HIV Env with strong CD8⁺ responses controlling early viral replication.     

Comparison of DNA vaccines producing HIV-1 Gag and LAMP/Gag chimera in rhesus macaques reveals antigen-specific T-cell responses with distinct phenotypes

Optimized DNA expression vectors encoding the native HIV-1 Gag or a fusion of Gag with the lysosomal membrane associated protein 1 (LAMP) were compared for immunogenicity upon intramuscular DNA delivery in rhesus macaques. Both vaccines elicited CD4⁺ T-cell responses, but with significant differences in the phenotype of the Gag-specific cells: the native Gag induced CD4⁺ responses with a phenotype of central memory-like T cells (CD28⁺ CD45RA⁻), whereas the LAMP/Gag chimera induced CD4⁺ responses with effector memory phenotype (CD28⁻ CD45RA⁻). Antigen-specific T cells producing both IFN-γ and TNFα were found in the animals receiving the native Gag, whereas the LAMP/Gag chimera induced humoral responses faster. These results demonstrate that modification of intracellular Gag trafficking results in the induction of distinct immune responses. Combinations of DNA vectors encoding both forms of antigen may be more potent in eliciting anti-HIV-1 immunity.     

Augmentation of SIV DNA vaccine-induced cellular immunity by targeting the 4-1BB costimulatory molecule

DNA vaccines are effective at inducing antigen-specific cellular immune responses. Approaches to improve these responses, however, are needed. We examined the effect of stimulating 4-1BB, an activation-inducible T-cell costimulatory receptor, by intravenously co-administering anti-human 4-1BB monoclonal antibody (mAb) in DNA-immunized cynomolgus macaques. Three groups of six cynomolgus macaques were immunized intramuscularly with a DNA vaccine encoding SIV Gag antigen (pSIVgag) at weeks 0, 4 and 8. At days 12, 15, and 19, six macaques received anti-4-1BB 4E9 mAb and six macaques received anti-4-1BB 10C7 mAb. Treatment with 10C7 mAb led to a significant augmentation of SIV Gag-specific IFN-gamma, granzyme B and perforin responses. Treatment with humanized 4E9 mAb also resulted in an enhancement of SIV Gag-specific cellular responses but the magnitude was lower compared to animals receiving 10C7 mAb. These responses persisted up to week 40 and were mostly mediated by CD8(+) T cells. Treatment with anti-4-1BB mAb was more effective in driving the CD8(+) T cells toward a more differentiated CCR7(-)/CD45RA(+) effector state. This study demonstrates that targeting the 4-1BB molecule in vivo results in an enhanced and long-lasting cellular immune response. 4-1BB stimulation may be a promising approach to enhance the effectiveness of DNA vaccines.     

A plasmid encoding the extracellular domain of CD40 ligand and Montanide™ GEL01 as adjuvants enhance the immunogenicity and the protection induced by a DNA vaccine against BoHV-1

DNA vaccines are capable of inducing humoral and cellular immunity, and are important to control bovine herpesvirus 1 (BoHV-1), an agent of the bovine respiratory disease complex. In previous work, a DNA plasmid that encodes a secreted form of BoHV-1 glycoprotein D (pCIgD) together with commercial adjuvants provided partial protection against viral challenge of bovines. In this work, we evaluate new molecules that could potentiate the DNA vaccine. We show that a plasmid encoding a soluble CD40 ligand (CD40L) and the adjuvant Montanide? GEL01 (GEL01) activate in vitro bovine afferent lymph dendritic cells (ALDCs). CD40L is a co-stimulating molecule, expressed transiently on activated CD4+ T cells and, to a lesser extent, on activated B cells and platelets. The interaction with its receptor, CD40, exerts effects on the presenting cells, triggering responses in the immune system. GEL01 was designed to improve transfection of DNA vaccines. We vaccinated cattle with: pCIgD; pCIgD-GEL01; pCIgD with GEL01 and CD40L plasmid (named pCIgD-CD40L-GEL01) or with pCIneo vaccines. The results show that CD40L plasmid with GEL01 improved the pCIgD DNA vaccine, increasing anti-BoHV-1 total IgGs, IgG1, IgG2 subclasses, and neutralizing antibodies in serum. After viral challenge, bovines vaccinated with pCIgD-GEL01-CD40L showed a significant decrease in viral excretion and clinical score. On the other hand, 80% of animals in group pCIgD-GEL01-CD40L presented specific anti-BoHV-1 IgG1 antibodies in nasal swabs. In addition, PBMCs from pCIgD-CD40L-GEL01 had the highest percentage of animals with a positive lymphoproliferative response against the virus and significant differences in the secretion of IFNγ and IL-4 by mononuclear cells, indicating the stimulation of the cellular immune response. Overall, the results demonstrate that a plasmid expressing CD40L associated with the adjuvant GEL01 improves the efficacy of a DNA vaccine against BoHV-1.     

Incorporation of 4-1BB ligand into an adenovirus vaccine vector increases the number of functional antigen-specific CD8 T cells and enhances the duration of protection against influenza-induced respiratory disease

T cell based influenza vaccines offer the potential for cross protective immunity to multiple clades of influenza virus. Here we explored the effect of increasing CD8 T cell responses during intranasal vaccination by incorporating a T cell costimulator, 4-1BBL. Inclusion of 4-1BBL in an influenza nucleoprotein (NP)-containing adenoviral vector increased the number of NP-specific CD8 T cells and lowered the vaccine dose required for short-term protection from influenza-induced disease in mice. At higher vaccine doses, the inclusion of 4-1BBL increased the duration of protection of mice from influenza-induced mortality. Bone marrow chimera experiments revealed that the major effects of 4-1BBL were directly on αβ T cells with minor additional effects through cells other than αβ T cells. The implications of these findings are that including 4-1BBL or adjuvants that induce 4-1BBL expression may be of benefit in a vaccine setting for enhancing the magnitude and duration of T cell responses to influenza virus.     

Robust Th1 cellular and humoral responses generated by the Yersinia pestis rF1-V subunit vaccine formulated to contain an agonist of the CD137 pathway do not translate into increased protection against pneumonic plague

Yersinia pestis is the causative agent of plague and is a re-emerging pathogen that also has the potential as a biological weapon, necessitating the development of a preventive vaccine. Despite intense efforts for the last several decades, there is currently not a vaccine approved by the FDA. The rF1-V vaccine adjuvanted with Alhydrogel is a lead candidate subunit vaccine for plague and generates a strong Th2-mediate humoral response with a modest Th1 cellular response. As immune protection against Y. pestis requires both humoral and Th1 cellular responses, modifying the rF1-V subunit vaccine formulation to include a robust inducer of Th1 responses may improve efficacy. Thus, we reformulated the subunit vaccine to include SA-4-1BBL, an agonist of the CD137 costimulatory pathway and a potent inducer of Th1 response, and assessed its protective efficacy against pneumonic plague. We herein show for the first time a sex bias in the prophylactic efficacy of the Alhydrogel adjuvanted rF1-V vaccine, with female mice showing better protection against pneumonic plague than male. The sex bias for protection was irrespective of the generation of comparable levels of rF1-V-specific antibody titers and Th1 cellular responses in both sexes. The subunit vaccine reformulated with SA-4-1BBL generated robust Th1 cellular and humoral responses. A prime-boost vaccination scheme involving prime with rF1-V + Alhydrogel and boost with the rF1-V + SA-4-1BBL provided protection in male mice against pneumonic plague. In marked contrast, prime and boost with rF1-V reformulated with both adjuvants resulted in the loss of protection against pneumonic plague, despite generating high levels of humoral and Th1 cellular responses. While unexpected, these findings demonstrate the complexity of immune mechanisms required for protection. Elucidating mechanisms responsible for these differences in protection will help to guide the development of better prophylactic subunit vaccines effective against pneumonic plague.     

Viral load and clinical disease enhancement associated with a lentivirus cytotoxic T lymphocyte vaccine regimen

Effective DNA-based vaccines against lentiviruses will likely induce CTL against conserved viral proteins. Equine infectious anemia virus (EIAV) infects horses worldwide, and serves as a useful model for lentiviral immune control. Although attenuated live EIAV vaccines have induced protective immune responses, DNA-based vaccines have not. In particular, DNA-based vaccines have had limited success in inducing CTL responses against intracellular pathogens in the horse. We hypothesized that priming with a codon-optimized plasmid encoding EIAV Gag p15/p26 with co-administration of a plasmid encoding an equine IL-2/IgG fusion protein as a molecular adjuvant, followed by boosting with a vaccinia vector expressing Gag p15/p26, would induce protective Gag-specific CTL responses. Although the regimen induced Gag-specific CTL in four of seven vaccinated horses, CTL were not detected until after the vaccinia boost, and protective effects were not observed in EIAV challenged vaccinates. Unexpectedly, vaccinates had significantly higher viral loads and more severe clinical disease, associated with the presence of vaccine-induced CTL. It was concluded that (1) further optimization of the timing and route of DNA immunization was needed for efficient CTL priming in vivo, (2) co-administration of the IL-2/IgG plasmid did not enhance CTL priming by the Gag p15/p26 plasmid, (3) vaccinia vectors are useful for lentivirus-specific CTL induction in the horse, (4) Gag-specific CTL alone are either insufficient or a more robust Gag-specific CTL response is needed to limit EIAV viremia and clinical disease, and (5) CTL-inducing vaccines lacking envelope immunogens can result in lentiviral disease enhancement. Although the mechanisms for enhancement associated with this vaccine regimen remain to be elucidated, these results have important implications for development of lentivirus T cell vaccines.     

Comparative ability of various plasmid-based cytokines and chemokines to adjuvant the activity of HIV plasmid DNA vaccines

The effectiveness of plasmid DNA (pDNA) vaccines can be improved by the co-delivery of plasmid-encoded molecular adjuvants. We evaluated pDNAs encoding GM-CSF, Flt-3L, IL-12 alone, or in combination, for their relative ability to serve as adjuvants to augment humoral and cell-mediated immune responses elicited by prototype pDNA vaccines. In Balb/c mice we found that co-administration of plasmid-based murine GM-CSF (pmGM-CSF), murine Flt-3L (pmFlt-3L) or murine IL-12 (pmIL-12) could markedly enhance the cell-mediated immune response elicited by an HIV-1 env pDNA vaccine. Plasmid mGM-CSF also augmented the immune response elicited by DNA vaccines expressing HIV-1 Gag and Nef-Tat-Vif. In addition, the use of pmGM-CSF as a vaccine adjuvant appeared to markedly increase antigen-specific proliferative responses and improved the quality of the resulting T-cell response by increasing the percentage of polyfunctional memory CD8(+) T cells. Co-delivery of pmFlt-3L with pmGM-CSF did not result in a further increase in adjuvant activity. However, the co-administration of pmGM-CSF with pmIL-12 did significantly enhance env-specific proliferative responses and vaccine efficacy in the murine vaccinia virus challenge model relative to mice immunized with the env pDNA vaccine adjuvanted with either pmGM-CSF or pmIL-12 alone. These data support the testing of pmGM-CSF and pmIL-12, used alone or in combination, as plasmid DNA vaccine adjuvants in future macaque challenge studies.     

SA-4-1BBL as the immunomodulatory component of a HPV-16 E7 protein based vaccine shows robust therapeutic efficacy in a mouse cervical cancer model

Cervical cancer is the leading cause of cancer-related deaths among women worldwide. Current prophylactic vaccines based on HPV (Human papillomavirus) late gene protein L1 are ineffective in therapeutic settings. Therefore, there is an acute need for the development of therapeutic vaccines for HPV associated cancers. The HPV E7 oncoprotein is expressed in cervical cancer and has been associated with the cellular transformation and maintenance of the transformed phenotype. As such, E7 protein represents an ideal target for the development of therapeutic subunit vaccines against cervical cancer. However, the low antigenicity of this protein may require potent adjuvants for therapeutic efficacy. We recently generated a novel chimeric form of the 4-1BBL costimulatory molecule engineered with core streptavidin (SA-4-1BBL) and demonstrated its safe and pleiotropic effects on various cells of the immune system. We herein tested the utility of SA-4-1BBL as the immunomodulatory component of HPV-16 E7 recombinant protein based therapeutic vaccine in the E7 expressing TC-1 tumor as a model of cervical cancer in mice. A single subcutaneous vaccination was effective in eradicating established tumors in approximately 70% of mice. The therapeutic efficacy of the vaccine was associated with robust primary and memory CD4⁺ and CD8⁺ T cell responses, Th1 cytokine response, infiltration of CD4⁺ and CD8⁺ T cells into the tumor, and enhanced NK cell killing. Importantly, NK cells played an important role in vaccine mediated therapy since their physical depletion compromised vaccine efficacy. Collectively, these data demonstrate the utility of SA-4-1BBL as a new class of multifunctional immunomodulator for the development of therapeutic vaccines against cancer and chronic infections.     

Dendritic cells infected by recombinant rabies virus vaccine vector expressing HIV-1 Gag are immunogenic even in the presence of vector-specific immunity

Dendritic cells (DC) are the most potent antigen presenting cells whose ability to interact with T cells, B cells and NK cells has led to their extensive use in vaccine design. Here, we designed a DC-based HIV-1 vaccine using an attenuated rabies virus vector expressing HIV-1 Gag (RIDC-Gag). To test this, BALB/c mice were immunized with RIDC-Gag, and the primary, secondary as well as humoral immune responses were monitored. Our results indicate that RIDC-Gag stimulated HIV-1 Gag-specific immune responses in mice. When challenged with vaccinia virus (VV) expressing HIV-1 Gag, they elicited a potent Gag-specific recall response characterized by CD8+ T cells expressing multiple cytokines that were capable of specifically lysing Gag-pulsed target cells. Moreover, RIDC-Gag also enhanced CD8+ T cell responses via a homologous prime-boost regimen. These results show that a DC-based vaccine using live RV is immunogenic and a potential candidate for a therapeutic HIV-1 vaccine.     

CRACC-targeting Fc-fusion protein induces activation of NK cells and DCs and improves T cell immune responses to antigenic targets

The CD2-like receptor activating cytotoxic cell (CRACC) receptor is a member of the SLAM family of receptors that are found on several types of immune cells. We previously demonstrated that increasing the abundance of the adaptor protein EAT-2 during vaccination enhanced innate and adaptive immune responses to vaccine antigens. Engagement of the CRACC receptor in the presence of the EAT-2 adaptor generally results in immune cell activation, while activating CRACC signaling in cells that lack EAT-2 adaptor inhibits their effector and regulatory functions. As EAT-2 is the only SAP adaptor that interacts with the CRACC receptor, we hypothesized that technologies that specifically modulate CRACC signaling during vaccination may also improve antigen specific adaptive immune responses. To test this hypothesis, we constructed a CRACC-targeting Fc fusion protein and included it in vaccination attempts. Indeed, mice co-vaccinated with the CRACC-Fc fusion protein and an adenovirus vaccine expressing the HIV-Gag protein had improved Gag-specific T cell responses, as compared to control mice. These responses are characterized by increased numbers of Gag-specific tetramer+ CD8+ T cells and increases in production of IFNγ, TNFα, and IL2, by Gag-specific CD8+ T cells. Moreover, our results revealed that use of the CRACC-Fc fusion protein enhances vaccine-elicited innate immune responses, as characterized by increased dendritic cells (DCs) maturation and IFNγ production from NK cells. This study highlights the importance of CRACC signaling during the induction of an immune response generally, and during vaccinations specifically, and also lends insight into the mechanisms underlying our prior results noting EAT-2-dependent improvements in vaccine efficacy.     

4-1BB ligand enhances tumor-specific immunity of poxvirus vaccines

PURPOSE: Recombinant poxvirus vaccines have been explored as tumor vaccines. The immunogenicity of these vaccines can be enhanced by co-expressing costimulatory molecules and tumor-associated antigens. While the B7-CD28 interaction has been most comprehensively investigated, other costimulatory molecules utilize different signaling pathways and might provide further cooperation in T cell priming and survival. 4-1BB (CD137) is a TNF family member and is critical for activation and long-term maintenance of primed T cells. This study was conducted to determine if a poxvirus expressing the ligand for 4-1BB (4-1BBL) could further improve the immune and therapeutic responses of a previously reported poxvirus vaccine expressing a triad of costimulatory molecules (B7.1, ICAM-1, and LFA-3). EXPERIMENTAL DESIGN: A recombinant vaccinia virus expressing 4-1BBL was generated and characterized in an in vitro infection system. This vaccine was then used alone or in combination with a vaccinia virus expressing CEA, B7.1, ICAM-1, and LFA-3 in CEA-transgenic mice bearing established MC38 tumors. Tumor growth and immune responses against CEA and other tumor-associated antigens were determined. The level of anti-apoptotic proteins in responding T cells was determined by flow cytometry on tetramer selected T cells. RESULTS: The combination of 4-1BBL with B7.1-based poxvirus vaccination resulted in significantly enhanced therapeutic effects against CEA-expressing tumors in a CEA-transgenic mouse model. This was associated with an increased level of CEA-specific CD4(+) and CD8(+) T cell responses, induction of antigen spreading to p53 and gp70, increased accumulation of CEA-specific T cells in the tumor microenvironment, and increased expression of bcl-X(L) and bcl-2 in CD4(+) and CD8(+) T cells in vaccinated mice. CONCLUSION: 4-1BBL cooperates with B7 in enhancing anti-tumor and immunologic responses in a recombinant poxvirus vaccine model. The inclusion of costimulatory molecules targeting distinct T cell signaling pathways provides a mechanism for enhancing the therapeutic effectiveness of tumor vaccines.     

Gene gun administration of therapeutic HPV DNA vaccination restores the efficacy of prolonged defrosted viral based vaccine

Freshly defrosted vaccines generate promising antitumor immunity by raising both robust CD8 and CD4 responses with a TC1/Th1-dominant cytokine profile. However, prolonged (overnight) defrosted Sindbis virus-E7/HSP70 priming and Vaccinia-E7/HSP70 booster in mouse model only elicited 20% long-term tumor-free survival in comparison with the fresh vaccines. The present study is to search the possible cause of its potency loss, and to evaluate the ability of pcDNA-E7/HSP70 DNA vaccination via gene gun in restoring the efficacy of E7-specific immune responses and antitumor properties. We used prolonged defrosted SINrep5-E7/HSP70 prime and defrostedVac-E7/HSP70 boost subcutaneously, and administered intradermally cluster (3-day interval) gene gun plasmid E7–HSP70DNA vaccine twice, and evaluated its ability to generate antigen-specific cytotoxic CD8+ T-cell responses using flow cytometry as well as antitumor responses using animal positron-emission tomography (PET) imaging. The prolonged defrosted vaccines showed a significant reduction in the infectivity and a significant decrease of CD8+ and CD4+ T-cells immune responses. Administration of cluster gene gun plasmid E7–HSP70DNA twice was also found to lead to restoration of immunity that elicits a full recovery of the antitumor efficacy of the prolonged defrosted vaccines. Our study suggested that adding cluster gene gun plasmid E7–HSP70DNA vaccine twice offered a simple solution in restoring the efficacy of the prime-boost vaccination with viral vectors and has potentially significant clinical applications.     

Enhanced cellular immune responses to SIV Gag by immunization with influenza and vaccinia virus recombinants

An effective vaccination strategy against human immunodeficiency virus type 1 (HIV-1) should include the induction of potent cellular immune responses against conserved HIV-1 antigens. We have generated five replication competent recombinant influenza viruses (rFlu/SIV Gag nos. 1-5) expressing different portions of Gag of simian immunodeficiency virus (SIV). Single intranasal immunizations in mice with each rFlu/SIV Gag viruses resulted in different degrees of protection against a challenge with recombinant vaccinia virus expressing SIV Gag. Immunized BALB/c mice had detectable CD8+ T cell responses specific for Gag peptide 185-199 when mice were vaccinated with rFlu/SIV Gag no. 3 virus, and for Gag peptides 281-295 and 285-299 when vaccinated with rFlu/SIV Gag no. 4 virus. Cellular immune responses against SIV Gag were further enhanced by a booster with a recombinant vaccinia virus expressing SIV Gag in both the spleen and local lymph node tissues, resulting in the induction of robust Gag-specific CD8+ T cell responses at both systemic and mucosal levels. We suggest that a prime-boost immunization regimen using recombinant influenza and vaccinia viruses expressing HIV Gag might represent an effective means to induce potent HIV-specific, protective CD8+ T cell responses.