ovalbumin and Vaccinia

A variety of strains of vaccinia virus (VACV) have been used as recombinant vaccine vectors with the aim of inducing robust CD8

Topics: Animals; Antigens, Viral; CD8-Positive T-Lymphocytes; Female; Genome, Viral; Immunization; Mice; Mice, Inbred C57BL; Ovalbumin; Peptide Fragments; Thymidine Kinase; Vaccinia; Vaccinia virus; Viral Envelope Proteins; Viral Proteins; Viral Vaccines

In contrast to responses against infectious challenge, T cell responses induced via adjuvanted subunit vaccination are dependent on interleukin-27 (IL-27). We show that subunit vaccine-elicited cellular responses are also dependent on IL-15, again in contrast to the infectious response. Early expression of interferon regulatory factor 4 (IRF4) was compromised in either IL-27- or IL-15-deficient environments after vaccination but not infection. Because IRF4 facilitates metabolic support of proliferating cells via aerobic glycolysis, we expected this form of metabolic activity to be reduced in the absence of IL-27 or IL-15 signaling after vaccination. Instead, metabolic flux analysis indicated that vaccine-elicited T cells used only mitochondrial function to support their clonal expansion. Loss of IL-27 or IL-15 signaling during vaccination resulted in a reduction in mitochondrial function, with no corresponding increase in aerobic glycolysis. Consistent with these observations, the T cell response to vaccination was unaffected by in vivo treatment with the glycolytic inhibitor 2-deoxyglucose, whereas the response to viral challenge was markedly lowered. Collectively, our data identify IL-27 and IL-15 as critical to vaccine-elicited T cell responses because of their capacity to fuel clonal expansion through a mitochondrial metabolic program previously thought only capable of supporting quiescent naïve and memory T cells.

Topics: Adjuvants, Immunologic; Aerobiosis; Allergens; Animals; Female; Glycolysis; Interleukin-15; Interleukins; Mice, Inbred C57BL; Mice, Transgenic; Orthomyxoviridae Infections; Ovalbumin; T-Lymphocytes; Vaccines, Subunit; Vaccinia

An elusive goal of cellular immune vaccines is the generation of large numbers of antigen-specific T cells in response to subunit immunization. A broad spectrum of cytokines and cell-surface costimulatory molecules are known to shape the programming, magnitude, and repertoire of T cells responding to vaccination. We show here that the majority of innate immune receptor agonist-based vaccine adjuvants unexpectedly depend on IL-27 for eliciting CD4(+) and CD8(+) T-cell responses. This is in sharp contrast to infectious challenge, which generates T-cell responses that are IL-27-independent. Mixed bone marrow chimera experiments demonstrate that IL-27 dependency is T cell-intrinsic, requiring T-cell expression of IL-27Rα. Further, we show that IL-27 dependency not only dictates the magnitude of vaccine-elicited T-cell responses but also is critical for the programming and persistence of high-affinity T cells to subunit immunization. Collectively, our data highlight the unexpected central importance of IL-27 in the generation of robust, high-affinity cellular immune responses to subunit immunization.

Topics: Adaptive Immunity; Adjuvants, Immunologic; Adoptive Transfer; Animals; Bacterial Vaccines; CD40 Antigens; Female; Immunologic Memory; Interleukins; Listeriosis; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Ovalbumin; Poly I-C; Radiation Chimera; Receptors, Cytokine; Receptors, Interleukin; Smallpox Vaccine; STAT Transcription Factors; T-Cell Antigen Receptor Specificity; T-Lymphocyte Subsets; Toll-Like Receptors; Transcription, Genetic; Vaccination; Vaccines, Subunit; Vaccinia

Topics: Allergens; Animals; Cytokines; Dermatitis, Allergic Contact; Eosinophils; Mice; Mice, Inbred BALB C; Mice, Knockout; Neutrophils; Ovalbumin; Receptors, Complement; RNA, Messenger; Vaccinia; Vaccinia virus; Viral Load

Modified vaccinia virus Ankara (MVA)-encoding antigens are considered as safe vaccine candidates for various infectious diseases in humans. Here, we investigated the immune-modulating properties of MVA-encoding ovalbumin (MVA-OVA) on the allergen-specific immune response.. The immune-modulating properties of MVA-OVA were investigated using GM-CSF-differentiated BMDCs from C57BL/6 mice. OVA expression upon MVA-OVA infection of BMDCs was monitored. Activation and maturation markers on viable MVA-OVA-infected mDCs were analyzed by flow cytometry. Secretion of INF-γ, IL-2, and IL-10 was determined in a co-culture of BMDCs infected with wtMVA or MVA-OVA and OVA-specific OT-I CD8(+) and OT-II CD4(+ ) T cells. BALB/c mice were vaccinated with wtMVA, MVA-OVA, or PBS, sensitized to OVA/alum and challenged with a diet containing chicken egg white. OVA-specific IgE, IgG1, and IgG2a and cytokine secretion from mesenteric lymph node (MLN) cells were analyzed. Body weight, body temperature, food uptake, intestinal inflammation, and health condition of mice were monitored.. Infection with wtMVA and MVA-OVA induced comparable activation of mDCs. MVA-OVA-infected BMDCs expressed OVA and induced enhanced IFN-γ and IL-2 secretion from OVA-specific CD8(+ ) T cells in comparison with OVA, wtMVA, or OVA plus wtMVA. Prophylactic vaccination with MVA-OVA significantly repressed OVA-specific IgE, whereas OVA-specific IgG2a was induced. MVA-OVA vaccination suppressed TH 2 cytokine production in MLN cells and prevented the onset of allergic symptoms and inflammation in a mouse model of OVA-induced intestinal allergy.. Modified vaccinia virus Ankara-ovalbumin (MVA-OVA) vaccination induces a strong OVA-specific TH 1- immune response, likely mediated by the induction of IFN-γ and IgG2a. Finally, MVA-based vaccines need to be evaluated for their therapeutic potential in established allergy models.

Topics: Allergens; Animals; Bone Marrow Transplantation; Cells, Cultured; Coculture Techniques; Dendritic Cells; Disease Models, Animal; Food Hypersensitivity; Immunotherapy, Adoptive; Inflammation; Intestinal Mucosa; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; Th1 Cells; Vaccines, Synthetic; Vaccinia; Vaccinia virus; Viral Vaccines

Efficient T-cell responses against recombinant antigens expressed by vaccinia virus vectors require expression of these antigens in the early phase of the virus replication cycle. The kinetics of recombinant gene expression in poxviruses are largely determined by the promoter chosen. We used the highly attenuated modified vaccinia virus Ankara (MVA) to determine the role of promoters in the induction of CD8 T-cell responses. We constructed MVA recombinants expressing either enhanced green fluorescent protein (EGFP) or chicken ovalbumin (OVA), each under the control of a hybrid early-late promoter (pHyb) containing five copies of a strong early element or the well-known early-late p7.5 or pS promoter for comparison. In primary or cultured cells, EGFP expression under the control of pHyb was detected within 30 min, as an immediate-early protein, and remained higher over the first 6 h of infection than p7.5- or pS-driven EGFP expression. Repeated immunizations of mice with recombinant MVA expressing OVA under the control of the pHyb promoter led to superior acute and memory CD8 T-cell responses compared to those to p7.5- and pS-driven OVA. Moreover, OVA expressed under the control of pHyb replaced the MVA-derived B8R protein as the immunodominant CD8 T-cell antigen after three or more immunizations. This is the first demonstration of an immediate-early neoantigen expressed by a poxviral vector resulting in superior induction of neoantigen-specific CD8 T-cell responses.

Topics: Animals; Antibodies, Viral; Antigens, Viral; Base Sequence; CD8 Antigens; CD8-Positive T-Lymphocytes; Cell Line; Cells, Cultured; Chick Embryo; Cricetinae; Female; Gene Expression; Genes, Immediate-Early; Genetic Vectors; Humans; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Ovalbumin; Promoter Regions, Genetic; Recombinant Fusion Proteins; Vaccinia; Vaccinia virus; Viral Vaccines

Although matured DC are capable of inducing effective primary and secondary immune responses in vivo, it is difficult to control the maturation and antigen loading in vitro. In this study, we show that ER-enriched microsomal membranes (microsomes) isolated from DC contain more peptide-receptive MHC I and II molecules than, and a similar level of costimulatory molecules to, their parental DC. After loading with defined antigenic peptides, the microsomes deliver antigenic peptide-MHC complexes (pMHC) to both CD4 and CD8 T cells effectively in vivo. The peptide-loaded microsomes accumulate in peripheral lymphoid organs and induce stronger immune responses than peptide-pulsed DC. The microsomal vaccines protect against acute viral infection. Our data demonstrate that peptide-MHC complexes armed microsomes from DC can be an important alternative to DC-based vaccines for protection from viral infection.

Topics: Animals; Antigen Presentation; Antigen-Presenting Cells; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Dendritic Cells; Endoplasmic Reticulum; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microsomes; Ovalbumin; Peptide Fragments; Spleen; Vaccinia; Viral Vaccines; Virus Diseases

We have analyzed the potential of virus-like particles (VLPs) from rabbit hemorrhagic disease virus (RHDV) as a delivery system for foreign T cell epitopes. To accomplish this goal, we generated chimeric RHDV-VLPs incorporating a CD8(+) T cell epitope (SIINFEKL) derived from chicken ovalbumin (OVA). The OVA epitope was inserted in the capsid protein (VP60) of RHDV at two different locations: 1) the N-terminus, predicted to be facing to the inner core of the VLPs, and 2) a novel insertion site predicted to be located within an exposed loop. Both constructions correctly assembled into VLPs. In vitro, the chimeric VLPs activated dendritic cells for TNF-alpha secretion and they were processed and presented to specific T cells. In vivo, mice immunized with the chimeric VLPs without adjuvant were able to induce specific cellular responses mediated by cytotoxic and memory T cells. More importantly, immunization with chimeric VLPs was able to resolve an infection by a recombinant vaccinia virus expressing OVA protein.

Topics: Adjuvants, Immunologic; Amino Acid Sequence; Animals; Caliciviridae Infections; CD8-Positive T-Lymphocytes; Chickens; Cytotoxicity Tests, Immunologic; Epitopes, T-Lymphocyte; Female; Hemorrhagic Disease Virus, Rabbit; Immunologic Memory; Injections, Intraperitoneal; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Ovalbumin; Reassortant Viruses; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Vaccination; Vaccinia; Vaccinia virus; Viral Vaccines; Virion

The magnitude of antigen-specific CD8+ T cell responses is not fixed but correlates with the severity of infection. Although by definition T cell response size is the product of both the capacity to recruit naïve T cells (clonal selection) and their subsequent proliferation (clonal expansion), it remains undefined how these two factors regulate antigen-specific T cell responses. We determined the relative contribution of recruitment and expansion by labeling naïve T cells with unique genetic tags and transferring them into mice. Under disparate infection conditions with different pathogens and doses, recruitment of antigen-specific T cells was near constant and close to complete. Thus, naïve T cell recruitment is highly efficient, and the magnitude of antigen-specific CD8+ T cell responses is primarily controlled by clonal expansion.

Topics: Adoptive Transfer; Ampicillin; Animals; Anti-Bacterial Agents; Antigens; Antigens, Bacterial; Antigens, Viral; CD8-Positive T-Lymphocytes; Dendritic Cells; Epitopes; Genes, T-Cell Receptor alpha; Genes, T-Cell Receptor beta; Influenza A virus; Listeriosis; Lymphocyte Activation; Lymphocyte Count; Mice; Mice, Inbred C57BL; Mice, Transgenic; Molecular Sequence Data; Orthomyxoviridae Infections; Ovalbumin; Receptors, Antigen, T-Cell, alpha-beta; Spleen; Vaccinia; Virus Diseases

CD8 T cell responses to vaccinia virus (VV) and a virus-encoded ovalbumin peptide (OVAP) epitope were examined using adoptively transferred OT-I T cells. The results demonstrate that upon intra-peritoneal challenge with ovalbumin-expressing VV (VV-OVAP), OT-I T cell proliferation occurs initially in lymph nodes and spleens followed by migration of the divided cells to the peritoneal cavity. Massive clonal expansion occurs in response to both the virus and the virus-encoded ovalbumin (OVA) epitope, as demonstrated using low numbers of adoptively transferred cells, and the responding OT-I cells display marked site-dependent functional heterogeneity with respect to IFN-gamma and tumor necrosis factor-alpha (TNF-alpha) production and granzyme B expression. OT-I cells responding to VV-OVAP develop the capacity to produce IFN-gamma in response to antigen as they proliferate and differentiate. In marked contrast, naive OT-I cells rapidly produce TNF-alpha upon antigen recognition, and this capacity declines as the cells proliferate in response to the virus, suggesting that this potent inflammatory cytokine may be important primarily during initiation of the response. At the peak of clonal expansion, a large fraction (30-60%) of the OT-I cells responding to the virus express high IL-7Ralpha levels, and the majority of these cells is subsequently lost. While high IL-7Ralpha expression may be necessary for a CD8 T cell to transition to memory, it is clearly not sufficient. Thus, OT-I cells responding to VV infection exhibit a high degree of heterogeneity within the responding population that differs depending on their anatomical location, despite the specificity and affinity of the TCR being identical on all of the cells.

Topics: Adoptive Transfer; Animals; CD8-Positive T-Lymphocytes; Cell Count; Cell Proliferation; Egg Proteins; Granzymes; Interferon-gamma; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; Peptide Fragments; Peritoneal Cavity; Receptors, Antigen, T-Cell; Receptors, Interleukin-7; Spleen; Thy-1 Antigens; Time Factors; Tumor Necrosis Factor-alpha; Vaccinia; Vaccinia virus

The nontoxic B subunit of Shiga toxin (STxB) targets in vivo Ag to dendritic cells that preferentially express the glycolipid Gb(3) receptor. After administration of STxB chemically coupled to OVA (STxB-OVA) or E7, a polypeptide derived from HPV, in mice, we showed that the addition of alpha-galactosylceramide (alpha-GalCer) resulted in a dramatic improvement of the STxB Ag delivery system, as reflected by the more powerful and longer lasting CD8(+) T cell response observed even at very low dose of immunogen (50 ng). This synergy was not found with other adjuvants (CpG, poly(I:C), IFN-alpha) also known to promote dendritic cell maturation. With respect to the possible mechanism explaining this synergy, mice immunized with alpha-GalCer presented in vivo the OVA(257-264)/K(b) complex more significantly and for longer period than mice vaccinated with STxB alone or mixed with other adjuvants. To test whether this vaccine could break tolerance against self Ag, OVA transgenic mice were immunized with STxB-OVA alone or mixed with alpha-GalCer. Although no CTL induction was observed after immunization of OVA transgenic mice with STxB-OVA, tetramer assay clearly detected specific anti-OVA CD8(+) T cells in 8 of 11 mice immunized with STxB-OVA combined with alpha-GalCer. In addition, vaccination with STxB-OVA and alpha-GalCer conferred strong protection against a challenge with vaccinia virus encoding OVA with virus titers in the ovaries reduced by 5 log compared with nonimmunized mice. STxB combined with alpha-GalCer therefore appears as a promising vaccine strategy to more successfully establish protective CD8(+) T cell memory against intracellular pathogens and tumors.

Topics: Animals; Autoantigens; Dendritic Cells; Drug Synergism; Galactosylceramides; Immune Tolerance; Mice; Mice, Transgenic; Oncogene Proteins, Viral; Ovalbumin; Papillomavirus E7 Proteins; Peptides; Shiga Toxins; T-Lymphocytes, Cytotoxic; Vaccines, Synthetic; Vaccinia; Vaccinia virus

The induction of chemokines by interferons might represent a link between innate and adaptive immunity. Whether these induced chemokines might be useful by themselves to induce an immune response is not known. We hypothesized that the interferon-inducible chemokine CXCL10 could stimulate dendritic cells (DC) to mature and cross-present exogenous antigen to T cells, resulting in a Th1-type immune response. We found that injecting mice with CXCL10 together with ovalbumin (OVA) as a test antigen was sufficient to produce functional OVA-specific T cells in 7 of 10 mice. Further, using only CXCL10 and a peptide antigen derived from vaccinia virus, we were able to induce peptide-specific cytotoxic T cells in 4 of 4 mice tested. These cytotoxic T cells protected 9 of 10 mice from subsequent infectious challenge with vaccinia virus. Unlike traditional adjuvants, no side effects were observed in any of the injected mice. We conclude that CXCL10 co-administration with a variety of antigens may represent a unique strategy of inducing a protective T cell response to a number of pathogens that merits further study.

Topics: Animals; Chemokine CXCL10; Chemokines, CXC; Cytotoxicity, Immunologic; Mice; Mice, Inbred C57BL; Ovalbumin; Skin Ulcer; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Vaccinia; Vaccinia virus; Viral Proteins

In mice with targeted disruption of the gene that encodes interleukin-6 (IL-6), greatly reduced numbers of immunoglobulin A (IgA)-producing cells were observed at mucosae and grossly deficient local antibody responses were recorded after mucosal challenge with either ovalbumin or vaccinia virus. The IgA response in the lungs was completely restored after intranasal infection with recombinant vaccinia viruses engineered to express IL-6. These findings demonstrate a critical role for IL-6 in vivo in the development of local IgA antibody responses and illustrate the effectiveness of vector-directed cytokine gene therapy.

Topics: Animals; Immunoglobulin A; Immunoglobulin G; Interleukin-6; Intestinal Mucosa; Lung; Lymph Nodes; Mesentery; Mice; Mucous Membrane; Mutation; Ovalbumin; Plasma Cells; Transfection; Vaccinia; Vaccinia virus