ISCOMATRIX adjuvant combines immune activation with antigen delivery to dendritic cells in vivo leading to effective cross-priming of CD8+ T cells

Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-γ production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8α(+) DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8α(+) DCs in VDLNs, whereas Langerin(+) DCs and CD8α(-) DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8α(+) DCs in vivo for efficient induction of CTL responses.     

Sustained peptide-specific gamma interferon T-cell response in rhesus macaques immunized with human immunodeficiency virus gag DNA vaccines

We examined the influence of dose and method of antigen delivery on the dynamics and durability of T-cell responses to candidate human immunodeficiency virus (HIV) vaccines. Codon-optimized sequences from the HIV gag gene were inserted into alternative DNA vaccine vectors to express the coding sequence with or without the tissue plasminogen activator leader sequence. We delivered the vaccines by intramuscular injection as plasmid DNA without adjuvant or as plasmid DNA formulated with a novel block copolymer adjuvant (CRL8623) and then monitored the ensuing T-cell responses by using a gamma interferon enzyme-linked immunospot assay. We demonstrated persistence of the cell-mediated immune (CMI) response in rhesus macaques for at least 18 months following a four-dose vaccination regimen. The plasmid vaccine, with or without CRL8623, was immunogenic in macaques; however, the form coadministered with adjuvant exhibited improved T-cell responses, with a bias toward more antigen-specific CD8(+) T cells. Finally, we examined the fine specificity of the T-cell response to the gag vaccines by testing the response of 23 vaccinated macaques to individual Gag 20-mer peptides. Collectively, the monkeys responded to 25 epitopes, and, on average, each monkey recognized a minimum of 2.7 epitopes. The results indicate that a broad and durable CMI response to HIV DNA vaccines can be induced in a relevant nonhuman primate model.     

A DNA vaccine encoding multiple HIV CD4 epitopes elicits vigorous polyfunctional, long-lived CD4+ and CD8+ T cell responses

T-cell based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection. CD4(+) T cells are important for the generation and maintenance of functional CD8(+) cytotoxic T cells. We have recently developed a DNA vaccine encoding 18 conserved multiple HLA-DR-binding HIV-1 CD4 epitopes (HIVBr18), capable of eliciting broad CD4(+) T cell responses in multiple HLA class II transgenic mice. Here, we evaluated the breadth and functional profile of HIVBr18-induced immune responses in BALB/c mice. Immunized mice displayed high-magnitude, broad CD4(+)/CD8(+) T cell responses, and 8/18 vaccine-encoded peptides were recognized. In addition, HIVBr18 immunization was able to induce polyfunctional CD4(+) and CD8(+) T cells that proliferate and produce any two cytokines (IFNγ/TNFα, IFNγ/IL-2 or TNFα/IL-2) simultaneously in response to HIV-1 peptides. For CD4(+) T cells exclusively, we also detected cells that proliferate and produce all three tested cytokines simultaneously (IFNγ/TNFα/IL-2). The vaccine also generated long-lived central and effector memory CD4(+) T cells, a desirable feature for T-cell based vaccines. By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4(+) T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8(+) T cells and antibody responses- elicited by other HIV immunogens.     

Characterization of hepatitis C virus core-specific immune responses primed in rhesus macaques by a nonclassical ISCOM vaccine

Current therapies for the treatment of hepatitis C virus (HCV) infection are only effective in a restricted number of patients. Cellular immune responses, particularly those mediated by CD8(+) CTLs, are thought to play a role in the control of infection and the response to antiviral therapies. Because the Core protein is the most conserved HCV protein among genotypes, we evaluated the ability of a Core prototype vaccine to prime cellular immune responses in rhesus macaques. Since there are serious concerns about using a genetic vaccine encoding for Core, this vaccine was a nonclassical ISCOM formulation in which the Core protein was adsorbed onto (not entrapped within) the ISCOMATRIX, resulting in approximately 1-microm particulates (as opposed to 40 nm for classical ISCOM formulations). We report that this Core-ISCOM prototype vaccine primed strong CD4(+) and CD8(+) T cell responses. Using intracellular staining for cytokines, we show that in immunized animals 0.30-0.71 and 0.32-2.21% of the circulating CD8(+) and CD4(+) T cells, respectively, were specific for naturally processed HCV Core peptides. Furthermore, this vaccine elicited a Th0-type response and induced a high titer of Abs against Core and long-lived cellular immune responses. Finally, we provide evidence that Core-ISCOM could serve as an adjuvant for the HCV envelope protein E1E2. Thus, these data provide evidence that Core-ISCOM is effective at inducing cellular and humoral immune responses in nonhuman primates.     

CD8+ T-cell priming regulated by cytokines of the innate immune system

Host protection against a variety of pathogens and tumours requires the efficient induction of CD8(+) T-cell responses. Yet, it has proven difficult to develop vaccines that effectively stimulate this type of cellular immunity. One well-defined obstacle is antigen accessibility to the MHC class I processing pathway. However, cytokines that are produced by cells of the innate immune system also have a key role in CD8(+) T-cell responses, by enhancing 'cross-presentation' and/or inducing CD8(+) T-cell priming and differentiation. Here, we discuss how innate cytokine responses regulate CD8(+) T-cell immunity, and argue that a greater understanding of these processes will be essential for effective tailoring of vaccine-induced cellular immune responses.     

Role of TLR4 in ethanol effects on innate and adaptive immune responses in peritoneal macrophages

Toll-like receptors (TLRs) play an important role in the innate immune response and these receptors link innate and adaptive responses. We have reported that ethanol modulates TLR4 receptors by activating or inhibiting its response. However, the role of TLRs in the effects of ethanol on the innate and adaptive responses during acute or chronic treatment is presently unknown. Peritoneal macrophages of wild-type and TLR4-deficient mice treated with acute ethanol (4?g?kg(-1), intraperitoneally) or chronic ethanol consumption (5 months) were used. Here we report how acute ethanol dose induces inflammatory cytokines (tumor necrosis factor-α, interleukin (IL)-1β, macrophage inflammatory protein 1α (MIP-1α), interferon β1 and IL-12β) and chemokines (monocyte chemoattractant protein -1α and MIP-1α), and upregulates major histocompatibility complexes class I and II (MHC-I and -II), but inhibits the activation of the costimulatory molecules (CD86 and CD40), leading to the suppression of the CD4(+) T-cell proliferation in the macrophages of wild-type mice. Chronic ethanol consumption downregulates the number of F4/80(+) cells expressing MHC-I and -II and decreases CD4(+) T-cell activation in wild-type mice. Interestingly, elimination of TLR4 abolishes the effects of ethanol on the innate and the adaptive inflammatory response induced by both ethanol treatments in macrophages. Taken together, our findings support the role of TLR4 in the effects of ethanol on the immune system, and suggest that alterations in the function of this receptor might modulate the immune response induced by alcohol abuse.     

Toll-like receptor adaptor molecules enhance DNA-raised adaptive immune responses against influenza and tumors through activation of innate immunity

Toll-like receptors (TLRs) recognize microbial components and trigger the signaling cascade that activates the innate and adaptive immunity. TLR adaptor molecules play a central role in this cascade; thus, we hypothesized that overexpression of TLR adaptor molecules could mimic infection without any microbial components. Dual-promoter plasmids that carry an antigen and a TLR adaptor molecule such as the Toll-interleukin-1 receptor domain-containing adaptor-inducing beta interferon (TRIF) or myeloid differentiation factor 88 (MyD88) were constructed and administered to mice to determine if these molecules can act as an adjuvant. A DNA vaccine incorporated with the MyD88 genetic adjuvant enhanced antigen-specific humoral immune responses, whereas that with the TRIF genetic adjuvant enhanced cellular immune responses. Incorporating the TRIF genetic adjuvant in a DNA vaccine targeting the influenza HA antigen or the tumor-associated antigen E7 conferred superior protection. These results indicate that TLR adaptor molecules can bridge innate and adaptive immunity and potentiate the effects of DNA vaccines against virus infection and tumors.     

Induction in humans of CD8+ and CD4+ T cell and antibody responses by sequential immunization with malaria DNA and recombinant protein

Vaccine-induced protection against diseases like malaria, AIDS, and cancer may require induction of Ag-specific CD8(+) and CD4(+) T cell and Ab responses in the same individual. In humans, a recombinant Plasmodium falciparum circumsporozoite protein (PfCSP) candidate vaccine, RTS,S/adjuvant system number 2A (AS02A), induces T cells and Abs, but no measurable CD8(+) T cells by CTL or short-term (ex vivo) IFN-gamma ELISPOT assays, and partial short-term protection. P. falciparum DNA vaccines elicit CD8(+) T cells by these assays, but no protection. We report that sequential immunization with a PfCSP DNA vaccine and RTS,S/AS02A induced PfCSP-specific Abs and Th1 CD4(+) T cells, and CD8(+) cytotoxic and Tc1 T cells. Depending upon the immunization regime, CD4(+) T cells were involved in both the induction and production phases of PfCSP-specific IFN-gamma responses, whereas, CD8(+) T cells were involved only in the production phase. IFN-gamma mRNA up-regulation was detected in both CD45RA(-) (CD45RO(+)) and CD45RA(+)CD4(+) and CD8(+) T cell populations after stimulation with PfCSP peptides. This finding suggests CD45RA(+) cells function as effector T cells. The induction in humans of the three primary Ag-specific adaptive immune responses establishes a strategy for developing immunization regimens against diseases in desperate need of vaccines.     

Vaccine-induced CD4+ T cell responses to MAGE-3 protein in lung cancer patients

MAGE-3 is the most commonly expressed cancer testis Ag and thus represents a prime target for cancer vaccines, despite infrequent natural occurrence of MAGE-3-specific immune responses in vivo. We report in this study the successful induction of Ab, CD8(+), and CD4(+) T cells in nonsmall cell lung cancer patients vaccinated with MAGE-3 recombinant protein. Two cohorts were analyzed: one receiving MAGE-3 protein alone, and one receiving MAGE-3 protein with adjuvant AS02B. Of nine patients in the first cohort, three developed marginal Ab titers and another one had a CD8(+) T cell response to HLA-A2-restricted peptide MAGE-3 271-279. In contrast, of eight patients from the second cohort vaccinated with MAGE-3 protein and adjuvant, seven developed high-titered Abs to MAGE-3, and four had a strong concomitant CD4(+) T cell response to HLA-DP4-restricted peptide 243-258. One patient simultaneously developed CD8(+) T cells to HLA-A1-restricted peptide 168-176. The novel monitoring methodology used in this MAGE-3 study establishes that protein vaccination induces clear CD4(+) T cell responses that correlate with Ab production. This development provides the framework for further evaluating integrated immune responses in vaccine settings and for optimizing these responses for clinical benefit.     

Induction of neutralizing antibodies and Th1-polarized and CD4-independent CD8+ T-cell responses following delivery of human immunodeficiency virus type 1 Tat protein by recombinant adenylate cyclase of Bordetella pertussis

HIV-Tat, a conserved protein playing a key role in the early life cycle of the human immunodeficiency virus (HIV) has been proposed as a potential AIDS vaccine. An HIV-Tat-based vaccine should elicit a broad, long-lasting, and neutralizing immune response. We have previously demonstrated that the adenylate cyclase (CyaA) from Bordetella pertussis targets dendritic cells and delivers CD8(+) and CD4(+) T-cell epitopes into the major histocompatibility complex class I and class II presentation pathways. We have also showed that CyaA induced specific and protective cytotoxic T cell responses in vivo. Here, we designed a prototype vaccine based on the HIV type 1 Tat delivered by CyaA (CyaA-E5-Tat) and tested its capacity to induce HIV-Tat-specific cellular as well as antibody responses. We showed that immunization of mice by CyaA-E5-Tat in the absence of adjuvant elicited strong and long-lasting neutralizing anti-Tat antibody responses more efficient than those obtained after immunization with Tat toxoid in aluminum hydroxide adjuvant. Analyses of the anti-Tat immunoglobulin G isotypes and the cytokine pattern showed that CyaA-E5-Tat induced a Th1-polarized immune response in contrast to the Th2-polarized immune responses obtained with the Tat toxoid. In addition, our data demonstrated that HIV-Tat-specific gamma interferon-producing CD8(+) T cells were generated after vaccination with CyaA-E5-Tat in a CD4(+) T-cell-independent manner. Based on these findings, CyaA-E5-Tat represents an attractive vaccine candidate for both preventive and therapeutic vaccination involving CyaA as an efficient nonreplicative vector for protein delivery.     

Vaccine-induced tumor-specific immunity despite severe B-cell depletion in mantle cell lymphoma

The role of B cells in T-cell priming is unclear, and the effects of B-cell depletion on immune responses to cancer vaccines are unknown. Although results from some mouse models suggest that B cells may inhibit induction of T cell-dependent immunity by competing with antigen-presenting cells for antigens, skewing T helper response toward a T helper 2 profile and/or inducing T-cell tolerance, results from others suggest that B cells are necessary for priming as well as generation of T-cell memory. We assessed immune responses to a well-characterized idiotype vaccine in individuals with severe B-cell depletion but normal T cells after CD20-specific antibody-based chemotherapy of mantle cell lymphoma in first remission. Humoral antigen- and tumor-specific responses were detectable but delayed, and they correlated with peripheral blood B-cell recovery. In contrast, vigorous CD4(+) and CD8(+) antitumor type I T-cell cytokine responses were induced in most individuals in the absence of circulating B cells. Analysis of relapsing tumors showed no mutations or change in expression of target antigen to explain escape from therapy. These results show that severe B-cell depletion does not impair T-cell priming in humans. Based on these results, it is justifiable to administer vaccines in the setting of B-cell depletion; however, vaccine boosts after B-cell recovery may be necessary for optimal humoral responses.     

Theiler's murine encephalomyelitis virus as a vaccine candidate for immunotherapy

The induction of sterilizing T-cell responses to tumors is a major goal in the development of T-cell vaccines for treating cancer. Although specific components of anti-viral CD8+ immunity are well characterized, we still lack the ability to mimic viral CD8+ T-cell responses in therapeutic settings for treating cancers. Infection with the picornavirus Theiler's murine encephalomyelitis virus (TMEV) induces a strong sterilizing CD8+ T-cell response. In the absence of sterilizing immunity, the virus causes a persistent infection. We capitalized on the ability of TMEV to induce strong cellular immunity even under conditions of immune deficiency by modifying the virus to evaluate its potential as a T-cell vaccine. The introduction of defined CD8+ T-cell epitopes into the leader sequence of the TMEV genome generates an attenuated vaccine strain that can efficiently drive CD8+ T-cell responses to the targeted antigen. This virus activates T-cells in a manner that is capable of inducing targeted tissue damage and glucose dysregulation in an adoptive T-cell transfer model of diabetes mellitus. As a therapeutic vaccine for the treatment of established melanoma, epitope-modified TMEV can induce strong cytotoxic T-cell responses and promote infiltration of the T-cells into established tumors, ultimately leading to a delay in tumor growth and improved survival of vaccinated animals. We propose that epitope-modified TMEV is an excellent candidate for further development as a human T-cell vaccine for use in immunotherapy.     

Vaccination with a DNA vaccine based on human PSCA and HSP70 adjuvant enhances the antigen-specific CD8+ T-cell response and inhibits the PSCA+ tumors growth in mice

BACKGROUND: DNA vaccines have been shown to be an effective approach to induce antigen-specific cellular and humoral immunity. However, the lower immune intensity in clinical trials limits the application of DNA vaccine. Here we intend to develop a new DNA vaccine based on prostate stem-cell antigen (PSCA), which has been suggested as a potential target for prostate cancer therapy, and enhance the DNA vaccine potency with heat shock proteins (HSPs) as adjuvant. METHODS: A series of DNA plasmids encoding human PSCA, human HSP70 and their conjugates was constructed and injected into male mice intramuscularly (i.m.). To evaluate the immune responses and therapeutic efficacy of these plasmids, major histocompatibility complex (MHC)-restricted PSCA and HSP70-specific epitopes were predicted and a mouse model with a human PSCA-expressing tumor was constructed. RESULTS: The result showed that mice vaccinated with PSCA-HSP plasmids generated the strongest PSCA-specific CD8+ T-cell immune response, but the CD4+ TH1 and TH2 cell immune responses were similar with those vaccinated with other HSP-adjuvant PSCA plasmids or only PSCA DNA. The immunity of HSP70 was also observed and the mice i.m. injected with PSCA+ HSP mixed plasmids generated the lowest anti-HSP antibodies. Furthermore, these vaccinations inhibited the growth of PSCA-expressing tumors and prolonged mouse survival. CONCLUSIONS: These observations emphasize and extend the potential of the human HSP70 gene as adjuvant for DNA vaccines, and the vaccine based on PSCA and HSP70 is of potential value for treating prostate cancer.     

Immunization with HIV-1 Gag protein conjugated to a TLR7/8 agonist results in the generation of HIV-1 Gag-specific Th1 and CD8+ T cell responses

One strategy to induce optimal cellular and humoral immune responses following immunization is to use vaccines or adjuvants that target dendritic cells and B cells. Activation of both cell types can be achieved using specific TLR ligands or agonists directed against their cognate receptor. In this study, we compared the ability of the TLR7/8 agonist R-848, which signals only via TLR7 in mice, with CpG oligodeoxynucleotides for their capacity to induce HIV-1 Gag-specific T cell and Ab responses when used as vaccine adjuvants with HIV-1 Gag protein in mice. Injection of R-848 and CpG oligodeoxynucleotides alone enhanced the innate immune responses in vivo as demonstrated by high serum levels of inflammatory cytokines, including IL-12p70 and IFN-alpha, and increased expression of CD80, CD86, and CD40 on CD11c(+) dendritic cells. By contrast, R-848 was a relatively poor adjuvant for inducing primary Th1 or CD8(+) T cell responses when administered with HIV-1 Gag protein. However, when a TLR7/8 agonist structurally and functionally similar to R-848 was conjugated to HIV-1 Gag protein both Th1 and CD8(+) T cells responses were elicited as determined by intracellular cytokine and tetramer staining. Moreover, within the population of HIV-1 Gag-specific CD8(+) CD62(low) cells, approximately 50% of cells expressed CD127, a marker shown to correlate with the capacity to develop into long-term memory cells. Overall, these data provide evidence that TLR7/8 agonists can be effective vaccine adjuvants for eliciting strong primary immune responses with a viral protein in vivo, provided vaccine delivery is optimized.     

Effects of type I interferons on the adjuvant properties of plasmid granulocyte-macrophage colony-stimulating factor in vivo

While administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) can induce the local recruitment of activated antigen-presenting cells at the site of vaccine inoculation, this cellular recruitment is associated with a paradoxical decrease in local vaccine antigen expression and vaccine-elicited CD8+ T-cell responses. To clarify why this cytokine administration does not potentiate immunization, we examined the recruited cells and expressed inflammatory mediators in muscles following intramuscular administration of plasmid GM-CSF in mice. While large numbers of dendritic cells and macrophages were attracted to the site of plasmid GM-CSF inoculation, high concentrations of type I interferons were also detected in the muscles. As type I interferons have been reported to damp foreign gene expression in vivo, we examined the possibility that these local innate mediators might decrease plasmid DNA expression and therefore the immunogenicity of plasmid DNA vaccines. In fact, we found that coadministration of an anti-beta interferon monoclonal antibody with the plasmid DNA immunogen and plasmid GM-CSF restored both the local antigen expression and the CD8+ T-cell immunogenicity of the vaccine. These data demonstrate that local innate immune responses can change the ability of vaccines to generate robust adaptive immunity.     

Induction of potent immune responses by cationic microparticles with adsorbed human immunodeficiency virus DNA vaccines

The effectiveness of cationic microparticles with adsorbed DNA at inducing immune responses was investigated in mice, guinea pigs, and rhesus macaques. Plasmid DNA vaccines encoding human immunodeficiency virus (HIV) Gag and Env adsorbed onto the surface of cationic poly(lactide-coglycolide) (PLG) microparticles were shown to be substantially more potent than corresponding naked DNA vaccines. In mice immunized with HIV gag DNA, adsorption onto PLG increased CD8(+) T-cell and antibody responses by approximately 100- and approximately 1,000-fold, respectively. In guinea pigs immunized with HIV env DNA adsorbed onto PLG, antibody responses showed a more rapid onset and achieved markedly higher enzyme-linked immunosorbent assay and neutralizing titers than in animals immunized with naked DNA. Further enhancement of antibody responses was observed in animals vaccinated with PLG/DNA microparticles formulated with aluminum phosphate. The magnitude of anti-Env antibody responses induced by PLG/DNA particles was equivalent to that induced by recombinant gp120 protein formulated with a strong adjuvant, MF-59. In guinea pigs immunized with a combination vaccine containing HIV env and HIV gag DNA plasmids on PLG microparticles, substantially superior antibody responses were induced against both components, as measured by onset, duration, and titer. Furthermore, PLG formulation overcame an apparent hyporesponsiveness of the env DNA component in the combination vaccine. Finally, preliminary data in rhesus macaques demonstrated a substantial enhancement of immune responses afforded by PLG/DNA. Therefore, formulation of DNA vaccines by adsorption onto PLG microparticles is a powerful means of increasing vaccine potency.     

A fusion inhibitor prevents spread of immunodeficiency viruses, but not activation of virus-specific T cells, by dendritic cells

Dendritic cells (DCs) play a key role in innate immune responses, and their interactions with T cells are critical for the induction of adaptive immunity. However, immunodeficiency viruses are efficiently captured by DCs and can be transmitted to and amplified in CD4(+) T cells, with potentially deleterious effects on the induction of immune responses. In DC-T-cell cocultures, contact with CD4(+), not CD8(+), T cells preferentially facilitated virus movement to and release at immature and mature DC-T-cell contact sites. This occurred within 5 min of DC-T-cell contact. While the fusion inhibitor T-1249 did not prevent virus capture by DCs or the release of viruses at the DC-T-cell contact points, it readily blocked virus transfer to and amplification in CD4(+) T cells. Higher doses of T-1249 were needed to block the more robust replication driven by mature DCs. Virus accumulated in DCs within T-1249-treated cocultures but these DCs were actually less infectious than DCs isolated from untreated cocultures. Importantly, T-1249 did not interfere with the stimulation of virus-specific CD4(+) and CD8(+) T-cell responses when present during virus-loading of DCs or for the time of the DC-T-cell coculture. These results provide clues to identifying strategies to prevent DC-driven virus amplification in CD4(+) T cells while maintaining virus-specific immunity, an objective critical in the development of microbicides and therapeutic vaccines.     

Vaccine-induced immunity in baboons by using DNA and replication-incompetent adenovirus type 5 vectors expressing a human immunodeficiency virus type 1 gag gene

The cellular immunogenicity of formulated plasmid DNA and replication-defective human adenovirus serotype 5 (Ad5) vaccine vectors expressing a codon-optimized human immunodeficiency virus type 1 gag gene was examined in baboons. The Ad5 vaccine was capable of inducing consistently strong, long-lived CD8(+)-biased T-cell responses and in vitro cytotoxic activities. The DNA vaccine-elicited immune responses were weaker than those elicited by the Ad5 vaccine and highly variable; formulation with chemical adjuvants led to moderate increases in the levels of Gag-specific T cells. Increasing the DNA-primed responses with booster doses of either Ad5 or modified vaccinia virus Ankara vaccines suggests a difference in the relative levels of cytotoxic and helper responses. The implications of these results are discussed.     

Multiple innate immune pathways contribute to the immunogenicity of recombinant adenovirus vaccine vectors

The innate immune pathways that contribute to the potent immunogenicity of recombinant adenovirus (rAd) vaccine vectors remain largely undefined. Previous studies assessing innate immunity triggered by vaccine vectors have largely focused on in vitro studies involving antigen-presenting cells and on early in vivo inflammatory responses. Here, we systematically explore the Toll-like receptor (TLR) signaling requirements for the generation of cellular immune responses by intramuscular immunization with common and alternative serotype rAd vectors in mice. Antigen-specific CD8(+) T-lymphocyte responses elicited by these rAd vectors were significantly diminished in MyD88(-/-) mice but not in TRIF(-/-) or TLR3(-/-) mice, suggesting the importance of MyD88-dependent TLR signaling. However, the absence of each individual TLR resulted in minimal to no effect on vaccine-elicited cellular immune responses. Moreover, responses were not diminished in IL-1R(-/-) or IL-18R(-/-) mice. These data suggest that rAd vectors engage multiple MyD88-dependent signaling pathways, none of which are individually critical; rather, they are integrated to contribute to the potent immunogenicity of rAd vectors. Stimulation of multiple innate immune mechanisms may prove a generalizable property of potent vaccines, and this strategy could be harnessed in the development of next-generation vaccine vectors and adjuvants.