MIS416, a non-toxic microparticle adjuvant derived from Propionibacterium acnes comprising immunostimulatory muramyl dipeptide and bacterial DNA promotes cross-priming and Th1 immunity

Propionibacterium acnes was modified using biochemical extraction methods generating a suspension of microparticles (MIS416) comprising a minimal cell wall skeleton rich in immunostimulatory crosslinked muramyl dipeptide repeats and native bacterial DNA fragments, each which have known adjuvant activity. In vitro studies demonstrated that MIS416 was readily internalized by human myeloid and plasmacytoid DC inducing cytokine secretion and cell activation/maturation. Vaccination studies in mice using OVA as a model antigen demonstrated that MIS416 acts as a Th1 adjuvant, promoting cross-priming of cytotoxic CD8⁺ T cell responses and enhanced anti-tumour immunity. Covalent attachment of OVA to MIS416 enabling simultaneous delivery of antigen and adjuvant to the antigen presentation system resulted in a dose-sparing vaccine formulation. Preclinical GLP toxicology studies demonstrated that MIS416 has a favorable safety profile in mouse and rabbit supporting its use in human vaccine formulations.     

CD8+ T cells specific to a single Yersinia pseudotuberculosis epitope restrict bacterial replication in the liver but fail to provide sterilizing immunity

CD8⁺ T cells use contact-dependent cytolysis of target cells to protect the host against intracellular pathogens. We have previously shown that CD8⁺ T cells and perforin are required to protect against the extracellular pathogen Yersinia pseudotuberculosis. Here we establish an experimental system where CD8⁺ T cells specific to a single model antigen are the only memory response present at time of challenge. Using mice immunized with a vaccine strain of Listeria monocytogenes that expresses secreted ovalbumin (Lm-OVA), we show that OVA-specific CD8⁺ T cells are generated and provide limited protection against challenge with virulent OVA⁺ Y. pseudotuberculosis. Perforin expression by OVA-specific CD8⁺ T cells was required, as Lm-OVA-immunized perforin-deficient mice showed higher bacterial burden as compared to Lm-OVA-immunized perforin-sufficient mice. Surprisingly, antigen-specific T cell protection waned over time, as Lm-OVA-immune mice eventually succumbed to Yersinia infection. Kinetic analysis of infection in mice with and without OVA-specific CD8⁺ T cells revealed that bacterial numbers increased sharply in OVA-naïve mice until death, while OVA-immune mice held bacterial burden to a lower level throughout the duration of illness until death. Clonal analysis of bacterial populations in OVA-naïve and OVA-immune mice at distinct time points revealed equivalent and severe bottle-neck effects for bacteria in both sets of mice immediately after intravenous challenge, demonstrating a dominant role for other aspects of the immune system regardless of CD8⁺ T cell status. These studies indicate that CD8⁺ T cells against a single antigen can restrict Y. pseudotuberculosis colonization in a perforin-dependent manner, but ultimately are insufficient in their ability to provide sterilizing immunity and protect against death.     

Induction of pulmonary mucosal immune responses with a protein vaccine targeted to the DEC-205/CD205 receptor

It is of great interest to develop a pneumonic plague vaccine that would induce combined humoral and cellular immunity in the lung. Here we investigate a novel approach based on targeting of dendritic cells using the DEC-205/CD205 receptor (DEC) via the intranasal route as way to improve mucosal cellular immunity to the vaccine. Intranasal administration of Yersinia pestis LcrV (V) protein fused to anti-DEC antibody together with poly IC as an adjuvant induced high frequencies of IFN-γ secreting CD4⁺ T cells in the airway and lung as well as pulmonary IgG and IgA antibodies. Anti-DEC:LcrV was more efficient to induce IFN-γ/TNF-α/IL-2 secreting polyfunctional CD4⁺ T cells when compared to non-targeted soluble protein vaccine. In addition, the intranasal route of immunization with anti-DEC:LcrV was associated with improved survival upon pulmonary challenge with the virulent CO92 Y. pestis. Taken together, these data indicate that targeting dendritic cells via the mucosal route is a potential new avenue for the development of a mucosal vaccine against pneumonic plague.     

Modulation of the CD4+ T cell response after acellular pertussis vaccination in the presence of TLR4 ligation

Whole cell pertussis (wP) vaccines are gradually being replaced by aluminum salt-adjuvanted acellular pertussis (aP) vaccines. These promote CD4⁺ T cell responses with a non-protective Th2 component, while protective immune mechanisms to B. pertussis may rather involve long-lived Th1/Th17 type CD4⁺ T cells. Here we asked whether addition of a non-toxic meningococcal LPS derivative, LpxL1, as adjuvant can favorably modulate the aP-induced pertussis-specific CD4⁺ T cell response in mice. To assess the effect of TLR4 ligation, Th type, quantity, and memory potential of pertussis-specific CD4⁺ T cells were determined at the single-cell level after aP and aP+LpxL1 vaccination using intracellular cytokine staining and MHC class II tetramers. Adding LpxL1 to the aP vaccine weakened the Th2 component and strengthened the Th1/Th17 component of the specific CD4⁺ T cell response. Notably, LpxL1 addition also induced higher frequencies of tetramer positive CD4⁺ T cells in draining lymph nodes or blood, depending on the phase after vaccination. Moreover, there was a net profit in the number of CD4⁺ T cells with a central memory phenotype, preferred for long-term immunity. Thus, adding a TLR4 ligand as adjuvant to a current aP vaccine was associated with a more favorable pertussis-specific CD4⁺ T cell response.     

Effective CD8 T cell priming and tumor protection by enterotoxin B subunit-conjugated peptides targeted to dendritic cells

In our previous studies we have shown that bacterial enterotoxin B subunits are effective vehicles to deliver antigen into the MHC class I processing route. Here we have used the non-toxic Escherichia coli heat labile enterotoxin B subunit (EtxB) conjugated to OVA peptide (EtxB–peptide) to address the impact on induction of specific CD8⁺ T cells in vivo. Although incubation of DCs with these EtxB–peptide conjugates as such did not induce DC maturation in vitro MHC class I antigen presentation was much more efficient as compared to peptide alone. Antigen presentation was further enhanced upon DC maturation with the TLR-4 ligand LPS. Injection of matured DCs incubated with EtxB–peptide conjugates lead to strong induction of OVA-specific CD8⁺ T lymphocytes and fully prevented the outgrowth of lethal B16 melanoma in wild type mice. Our data demonstrate that bacterial non-toxic B subunit–peptide conjugates are potent vaccine vehicles for induction of protective CD8⁺ T cell responses.     

Skin vaccination with live virus vectored microneedle arrays induce long lived CD8+ T cell memory

A simple dissolvable microneedle array (MA) platform has emerged as a promising technology for vaccine delivery, due to needle-free injection with a formulation that preserves the immunogenicity of live viral vectored vaccines dried in the MA matrix. While recent studies have focused largely on design parameters optimized to induce primary CD8⁺ T cell responses, the hallmark of a vaccine is synonymous with engendering long-lasting memory. Here, we address the capacity of dried MA vaccination to programme phenotypic markers indicative of effector/memory CD8⁺ T cell subsets and also responsiveness to recall antigen benchmarked against conventional intradermal (ID) injection. We show that despite a slightly lower frequency of dividing T cell receptor transgenic CD8⁺ T cells in secondary lymphoid tissue at an early time point, the absolute number of CD8⁺ T cells expressing an effector memory (CD62L⁻CD127⁺) and central memory (CD62L⁺CD127⁺) phenotype during peak expansion were comparable after MA and ID vaccination with a recombinant human adenovirus type 5 vector (AdHu5) encoding HIV-1 gag. Similarly, both vaccination routes generated CD8⁺ memory T cell subsets detected in draining LNs for at least two years post-vaccination capable of responding to secondary antigen. These data suggest that CD8⁺ T cell effector/memory generation and long-term memory is largely unaffected by physical differences in vaccine delivery to the skin via dried MA or ID suspension.     

CD11a and CD49d enhance the detection of antigen-specific T cells following human vaccination

BackgroundDetermining the efficacy of human vaccines that induce antigen-specific protective CD4 T cell responses against pathogens can be particularly challenging to evaluate. Surface expression of CD11a and CD49d has been shown to identify antigen-specific CD4 T cells against viral pathogens in mice. We hypothesized that CD11a and CD49d would also serve as markers of human antigen-specific T cells responding to vaccination.MethodsA phase I vaccine trial enabled us to evaluate a novel gating strategy based on surface expression of CD11a and CD49d as a means of detecting antigen-specific, cytokine producing CD4 and CD8 T cells induced after vaccination of naïve individuals against leishmaniasis. Three study groups received LEISH-F3 recombinant protein combined with either squalene oil-in-water emulsion (SE) alone, SE with the synthetic TLR-4 ligand glucopyranosyl lipid adjuvant (GLA-SE), or SE with Salmonella minnesota-derived monophosphoryl lipid A (MPL-SE). Individuals were given 3 vaccine doses, on days 0, 28 and 168.ResultsStarting after the first vaccine dose, the frequency of both CD11a^hiCD49d⁺ CD4 and CD11a^hiCD49d⁺ CD8 T cells significantly increased over time throughout the 24-week trial. To confirm the role of CD11a^hiCD49d⁺ expression in the identification of the antigen-specific T cells, cytokine production was measured following LEISH-F3 stimulation. All of the IFN-γ, TNF-α, and IL-2 producing cells were found within the CD11a^hiCD49d⁺ population.ConclusionsOur results suggest that the change in the frequency of CD11a^hiCD49d⁺ T cells can be used to track antigen-specific CD4 and CD8 T cell responses following T cell-targeted vaccination.     

Evaluation of hyaluronic acid-based combination adjuvant containing monophosphoryl lipid A and aluminum salt for hepatitis B vaccine

Here, monophosphoryl lipid A (MPLA) and aluminum salt (Alum) were introduced into a hyaluronic acid (HA)-based combination vaccine adjuvant for hepatitis B vaccine (HBV). Although Alum is a well-known hepatitis B vaccine adjuvant that induces an enhanced humoral immune response, it cannot induce the cellular immune responses. On the other hand, MPLA has been generally reported to promote IFN-γ production via antigen-specific CD4⁺ T cells, but it is not water soluble as a result of its long hydrophobic alkyl chains. To this end, water insoluble MPLA could be solubilized in an aqueous solution with the help of HA, which contains many carboxyl and hydroxyl groups that can be used to attach to the hydroxyl head groups of MPLA via hydrogen bonds. Three groups of mice were treated with either hepatitis B surface antigen (HBsAg) alone, HBsAg_Alum complex, or HBsAg_Alum_MPLA/HA complex. The group immunized with the HBsAg_Alum_MPLA/HA complex exhibited a high increase in cellular immune response as well as in humoral immune response relative to the other two groups. The antibody, cytokine and T cell levels were most elevated in the group of mice immunized with HBsAg_Alum_MPLA/HA complex, even at a 1 μg/mice dose, and the magnitude was still maintained even after 8 weeks. Specifically, the antibody value was 120 times larger in mice vaccinated with HBsAg_Alum_MPLA/HA complex than in mice vaccinated with HBsAg_Alum complex designed similar to commercially available hepatitis B vaccine, Engerix B. The cytokine and T cell proliferation levels were 2 times and 6 times larger in mice adjuvanted with HBsAg_Alum_MPLA/HA complex than in those vaccinated with HBsAg_Alum. The results therefore indicate that incorporating MPLA and Alum with HA can be a potent strategy to increase both the magnitude and the persistence of HBsAg-specific immune responses to protect hosts against hepatitis B virus infection.     

Effect of a combination DNA vaccine for the prevention and therapy of Trypanosoma cruzi infection in mice: Role of CD4 and CD8 T cells

Chagas disease is a major public health problem, with about 10 million infected people, and DNA vaccines are a promising alternative for the control of Trypanosoma cruzi, the causing agent of the disease. We tested here a new DNA vaccine encoding a combination of two leading parasite antigens, TSA-1 and Tc24, for the prevention and therapy of T. cruzi infection. Immunized Balb/c mice challenged by T. cruzi presented a significantly lower parasitemia and inflammatory cell density in the heart compared to control mice. Similarly, the therapeutic administration of the DNA vaccine was able to significantly reduce the parasitemia and inflammatory reaction in acutely infected Balb/c and C57BL/6 mice, and reduced cardiac tissue inflammation in chronically infected ICR mice. Therapeutic vaccination induced a marked increase in parasite-specific IFNγ producing CD4⁺ and CD8⁺ T cells in the spleen as well as an increase in CD4⁺ and CD8⁺ T cells in the infected cardiac tissue. In addition, some effect of the DNA vaccine could still be observed in CD4-knockout C57BL/6 mice, which presented a lower parasitemia and inflammatory cell density, but not in CD8-deficient mice, in which the vaccine had no effect. These results indicate that the activation of CD8⁺ T cells plays a major role in the control of the infection by the therapeutic DNA vaccine, and to a somewhat lesser extent CD4⁺ T cells. This observation opens interesting perspectives for the potentiation of this DNA vaccine candidate by including additional CD8⁺ T cell antigens/epitopes in future vaccine formulations.     

The adjuvancy of OX40 ligand (CD252) on an HIV-1 canarypox vaccine

The immunogenicity of current human immunodeficiency virus-1 (HIV-1) canarypox vaccines is weak and needs to be improved. Ligation of OX40 (CD134), a member of tumor necrosis factor receptor superfamily (TNFRSF), by its ligand OX40L (CD252), a tumor necrosis factor superfamily (TNFSF) molecule, has been demonstrated to provide a pivotal costimulatory signal to enhance CD4⁺ T cell help of humoral and cytotoxic T cell immune responses. The present study examined whether an OX40L-expressing vector could boost the immunogenicity of the HIV-1 canarypox vaccine, vCP1452, in mice. Co-immunization of mice with OX40L-expressing canarypox and vCP1452 augmented HIV-1 specific CD8⁺ T cell responses in terms of frequency and cytokine expression. OX40L-expressing canarypox enhanced the frequency of antigen specific CD8⁺ T cells with an effector (CD127⁻CD62L⁻) phenotype, which was associated with an ex vivo expansion of HIV-1 specific CD4⁺ T cells. This was in contrast to our previous work in which a CD40L-expressing construct preferentially enhanced antigen specific memory responses [Liu J, Yu Q, Stone GW, Yue FY, Ngai N, Jones RB, et al. CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in vitro expansion of viral specific CD8+ T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals. Vaccine 2008;26(32):4062–72]. Surprisingly, OX40L did not enhance antibody responses elicited by the HIV-1 canarypox vaccine. We saw no added benefit by combining OX40L and CD40L vectors as an adjuvant strategy for vCP1452. Our results indicate that, similar to CD40L, canarypox vectors expressing OX40L can enhance the cellular but not humoral immunogenicity of HIV-1 canarypox vaccines. In summary, our findings show that OX40L can be used as a molecular adjuvant to enhance T cell immune responses.     

Intranasal boosting with MVA encoding secreted mycobacterial proteins Ag85A and ESAT-6 generates strong pulmonary immune responses and protection against M. tuberculosis in mice given BCG as neonates

Bacille-Calmette-Guerin (BCG) has variable efficacy as an adult tuberculosis (TB) vaccine but can reduce the incidence and severity of TB infection in humans. We have engineered modified vaccinia Ankara (MVA) strain vaccine constructs to express the secreted mycobacterial proteins Ag85A and ESAT-6 (MVA-AE) and evaluated their immunogenicity and protective efficacy as mucosal booster vaccines for BCG given subcutaneously in early life. Intranasal delivery of MVA-AE to young adult mice induced CD4⁺ and CD8⁺ T cell responses to both Ag85A and ESAT-6 in lung mucosae. These responses were markedly enhanced in mice that had been primed neonatally with BCG prior to intranasal MVA-AE immunization (BCG/MVA-AE), as evidenced by numbers of pulmonary Ag85A-, ESAT-6-, and PPD-specific CD4⁺ and CD8⁺ T cells and by their capacity to secrete multiple antimicrobial factors, including IFNγ, IL-2 and IL-17. Moreover, MVA-AE boosting generated multifunctional lung CD4⁺ T cells responding to ESAT-6, which were not, as expected, detected in control mice given BCG, and elevated Ag85A-specific circulating antibody responses. After aerosol challenge with M. tuberculosis H37Rv (Mtb), the BCG/MVA-AE group had significantly reduced mycobacterial burden in the lungs, compared with either BCG primed mice boosted with control MVA or mice given only BCG. These data indicate that intranasal delivery of MVA-AE can boost BCG-induced Th1 and Th17-based immunity locally in the lungs and improve the protective efficacy of neonatally-administered BCG against M. tuberculosis infection.     

CD8 T cell adjuvant effects of Salmonella FliCd flagellin in live vaccine vectors or as purified protein

Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2^d-restricted CD8⁺ T cell-specific epitope (CS_280–288) derived from the Plasmodium yoelii circumsporozoite (CS) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS_280–288 peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS_280–288 peptide. The results showed that CS_280–288-specific cytotoxic CD8⁺ T cells were primed when BALB/c mice were orally inoculated with the expressing the CS_280–288 epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS_280–288 peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8⁺ T cell responses without the need of a heterologous booster immunization. The CD8⁺ T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c⁺ dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations.     

Recombinant pro-apoptotic Mycobacterium tuberculosis generates CD8 T cell responses against human immunodeficiency virus type 1 Env and M. tuberculosis in neonatal mice

Mycobacterium bovis BCG is an attractive vaccine vector against breast milk HIV transmission because it elicits Th1-type responses in newborns. However, BCG causes disease in HIV-infected infants. Genetically attenuated Mycobacterium tuberculosis (Mtb) mutants represent a safer alternative for immunocompromised populations. In the current study, we compared the immunogenicity in mice of three different recombinant attenuated Mtb strains expressing an HIV envelope (Env) antigen construct. Two of these strains (ΔlysA ΔpanCD Mtb and ΔRD1 ΔpanCD Mtb) failed to induce significant levels of HIV Env-specific CD8⁺ T cell responses. In striking contrast, an HIV-1 Env-expressing attenuated ΔlysA Mtb containing a deletion in secA2, which encodes a virulence-related secretion system involved in evading adaptive immunity, generated consistently measurable Env-specific CD8⁺ T cell responses that were significantly greater than those observed after immunization with BCG expressing HIV Env. Similarly, another strain of ΔlysA ΔsecA2 Mtb expressing SIV Gag induced Gag- and Mtb-specific CD8⁺ T cells producing perforin or IFNγ, and Gag-specific CD4⁺ T cells producing IFNγ within 3 weeks after immunization in adult mice; in addition, IFNγ-producing Gag-specific CD8⁺ T cells and Mtb-specific CD4⁺ T cells were observed in neonatal mice within 1 week of immunization. We conclude that ΔlysA ΔsecA2 Mtb is a promising vaccine platform to construct a safe combination HIV-TB vaccine for use in neonates.     

Profound CD8+ T cell immunity elicited by sequential daily immunization with exogenous antigen plus the TLR3 agonist poly(I:C)

The development of vaccines that elicit robust CD8⁺ T cell immunity has long been a subject of intense investigation. Although whole exogenous protein has not historically been considered as useful for eliciting CD8⁺ T cell immunity, we report herein that whole, protein antigen is capable of eliciting profound levels of CD8⁺ T cell immunity if it is administered via repeated, daily subcutaneous immunization in combination with the TLR3 agonist poly(I:C). Mice immunized for four consecutive days with 100 μg of either whole exogenous OVA or whole HPV16 E7 protein combined with 10 μg of poly(I:C) mounted remarkable antigen-specific CD8⁺ T cell responses as measured by tetramer staining and ELISPOT analysis of splenocytes and peripheral blood, with up to 30% of peripheral CD8⁺ T cells being antigen specific within 7-8 days of vaccination. CD8⁺ T cell immunity elicited using this vaccination approach was critically dependent upon cross presentation, as either whole protein or long synthetic peptides were highly effective immunogens whereas minimal peptide epitopes were not. Vaccine-induced CD8⁺ T cells were also able to regress large, established tumors in vivo. Together these data suggest that ‘cluster’ vaccination with exogenous antigen combined with TLR3 agonist may constitute a profoundly important advancement in therapeutic vaccine design.     

Brucella abortus Omp19 recombinant protein subcutaneously co-delivered with an antigen enhances antigen-specific T helper 1 memory responses and induces protection against parasite challenge

The discovery of effective adjuvants for many vaccines especially those with limited commercial appeal, such as vaccines to poverty-related diseases, is required. In this work, we demonstrated that subcutaneous co-administration of mice with the outer membrane protein U-Omp19 from Brucella spp. plus OVA as antigen (Ag) increases Ag-specific T cell proliferation and T helper (Th) 1 immune responses in vitro and in vivo. U-Omp19 treated dendritic cells promote IFN-γ production by specific CD4⁺ T cells and increases T cell proliferation. U-Omp19 co-administration induces the production of Ag specific effector memory T cell populations (CD4⁺ CD44^high CD62L^low T cells). Finally, subcutaneous co-administration of U-Omp19 with Trypanosoma cruzi Ags confers protection against virulent parasite challenge, reducing parasitemia and weight loss while increasing mice survival. These results indicate that the bacterial protein U-Omp19 when delivered subcutaneously could be a suitable component of vaccine formulations against infectious diseases requiring Th1 immune responses.     

Intranasal c-di-GMP-adjuvanted plant-derived H5 influenza vaccine induces multifunctional Th1 CD4+ cells and strong mucosal and systemic antibody responses in mice

Vaccination is the best available measure of limiting the impact of the next influenza pandemic. Ideally, a candidate pandemic influenza vaccine should be easy to administer and should elicit strong mucosal and systemic immune responses. Production of influenza subunit antigen in transient plant expression systems is an alternative to overcome the bottleneck in vaccine supply during influenza pandemic. Furthermore, a needle-free intranasal influenza vaccine is an attractive approach, which may provide immunity at the portal of virus entry.The present study investigated the detailed humoral and cellular immune responses in mice vaccinated intranasally or intramuscularly with plant-derived influenza H5N1 (A/Anhui/1/05) antigen alone or formulated with bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) as adjuvant. The use of c-di-GMP as intramuscular adjuvant did not enhance the immune response to plant-derived influenza H5 antigen. However, intranasal c-di-GMP-adjuvanted vaccine induced strong mucosal and systemic humoral immune responses. Additionally, the intranasal vaccine elicited a balanced Th1/Th2 profile and, most importantly, high frequencies of multifunctional Th1 CD4⁺ cells. Our results highlight that c-di-GMP is a promising mucosal adjuvant for pandemic influenza vaccine development.     

Meningococcal PorB induces a robust and diverse antigen specific T cell response as a vaccine adjuvant

Vaccines formulated with adjuvant have been effective against numerous infectious diseases, almost always due to induction of functional antibodies that recognizes the pathogen of interest. There is an unmet clinical need for vaccine adjuvants that induce T cells responses to potentially enhance protection against malignancies and intracellular pathogens, where a humoral response, alone, may not be adequate for protection. In this study, we demonstrate that a TLR2 ligand-based adjuvant, meningococcal PorB, has broad immunostimulatory activity with the ability to induce a robust and diverse vaccine antigen specific T cell response. We demonstrate that a vaccine formulated with PorB admixed with ovalbumin induces a wide variety of antigen specific antibody subclasses and effector molecules (MIG, MCP-1, IP-10, MIP-1α, KC & IL-2) with known roles for inducing T cell responses, along with elevated levels of Th1 and Th2 type cytokines upon antigen stimulation. We confirmed production of these cytokines by examining the antigen-specific T cells induced by PorB in vivo. After two immunizations with vaccine formulated with PorB/OVA, antigen-specific CD4 and CD8 T cells were significantly increased in numbers and produced IL-4 or IFN-γ upon ex vivo antigen re-stimulation. Finally, in a Listeria mouse infection model, vaccine formulated with PorB significantly reduced the bacterial burden upon a low dose infection and increased survival upon a high dose infection with recombinant Listeria monocytogenes engineered to express OVA (rLmOVA), a pathogen that requires OVA-antigen specific cytotoxic CD8 T cells for clearance. In summary, PorB is able to induce antigen specific broad B and T cell responses, illustrating its potential as a potent and new vaccine adjuvant.     

Anti-glioma effect of intracranial vaccination with tumor cell lysate plus flagellin in mice

The adjuvant effects of flagellin on regulation of immune response have been proved; whether flagellin could assist tumor cell lysate (TCL) to enhance anti-glioma immunity remains to be investigated. This study tests a hypothesis that therapeuticly intracranial administration with flagellin plus TCL enhances the effects of specific immunotherapy on glioma in mice. In this study, GL261 cells were transferred into C57BL/6 mice and the GL261-bearing mice were subcutaneously or intracranially inoculated with flagellin plus TCL, flagellin, TCL or saline. Our results showed that prophylacticly subcutaneous administration with TCL and flagellin could induce potent cytotoxic T lymphocyte (CTL) and prolong the survival of GL261-bearing mice significantly, but therapeuticly subcutaneous administration failed to. However, therapeuticly intracranial administration of TCL plus flagellin could prolong the survival. Moreover, intracranial administration of flagellin could recruit CD4⁺ T cells and CD8⁺ T cells to brain tissues, induce proliferation of natural killer (NK) cells, CD4⁺ T cells and CD8⁺ T cells in peripheral blood mononuclear cells and induce to splenomegaly. The results suggested that flagellin could be acted as an efficient adjuvant for TCL based vaccine.     

BCG vaccination-induced long-lasting control of Mycobacterium tuberculosis correlates with the accumulation of a novel population of CD4+IL-17+TNF+IL-2+ T cells

Mycobacterium bovis Bacille Calmette-Guerin (BCG) is the only vaccine in use to prevent Mycobacterium tuberculosis (Mtb) infection. Here we analyzed the protective efficacy of BCG against Mtb challenges 21 or 120 days after vaccination. Only after 120 days post-vaccination were mice able to efficiently induce early Mtb growth arrest and maintain long-lasting control of Mtb. This protection correlated with the accumulation of CD4⁺ T cells expressing IL-17⁺TNF⁺IL-2⁺. In contrast, mice challenged with Mtb 21 days after BCG vaccination exhibited only a mild and transient protection, associated with the accumulation of CD4⁺ T cells that were mostly IFN-γ⁺TNF⁺ and to a lesser extent IFN-γ⁺TNF⁺IL-2⁺. These data suggest that the memory response generated by BCG vaccination is functionally distinct depending upon the temporal proximity to BCG vaccination. Understanding how these responses are generated and maintained is critical for the development of novel vaccination strategies against tuberculosis.     

A Mage3/Heat Shock Protein70 DNA vaccine induces both innate and adaptive immune responses for the antitumor activity

Heat shock proteins (HSPs) are highly effective and versatile molecules in promoting antitumor immune responses. We tested whether a HSP-based DNA vaccine can induce effective immune response against Mage3, a cancer testis (CT) antigen frequently expressed in many human tumors, thereby controlling the Mage3-expressing tumor. The vaccine was constructed by linking human inducible HSP70 to the C-terminus of a modified Mage3 gene (sMage3) that was attached at its N-terminus with the signal leader sequence of the human RANTES for releasing the expressed fusion protein from the transduced cells. Intramuscular injection of sMage3Hsp DNA induced CD4⁺/CD8⁺ T cell and antibody responses. Vaccination with sMage3Hsp DNA was more effective in inhibiting Mage3-expressing TC-1 tumors. When we dissected the antitumor activity of CD4⁺ and CD8⁺ T cells by immunizing CD4⁺ and CD8⁺ knockout mice with sMage3Hsp DNA, we found that both CD8⁺ T and CD4⁺ T cells played a role in control of inoculated tumor, but did not constitute the whole of immune protection in the prophylactic immunization. Instead, depletion of natural killer (NK) cells led to a major loss of antitumor activity in the immunized mice. These results indicate that the HSP-based Mage3 DNA vaccine can more effectively inhibit tumor growth by inducing both the innate immune responses and Mage3-specific adaptive immune responses via the Hsp-associated adjuvant function.