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Tuberculosis remains a major public health hazard worldwide, with neonates and young infants potentially more susceptible to infection than adults. BCG, the only vaccine currently available, provides some protection against tuberculous meningitis in children but variable efficacy in adults, and is not safe to use in immune compromised individuals. A safe and effective vaccine that could be given early in life, and that could also potentiate subsequent booster immunization, would represent a significant advance. To test this proposition, we have generated gene-based vaccine vectors expressing Ag85B from Mycobacterium tuberculosis (Mtb) and designed experiments to test their immunogenicity and protective efficacy particularly when given in heterologous prime-boost combination, with the initial DNA vaccine component given soon after birth. Intradermal delivery of DNA vaccines elicited Th1-based immune responses against Ag85B in neonatal mice but did not protect them from subsequent aerosol challenge with virulent Mtb H37Rv. Recombinant adenovirus vectors encoding Ag85B, given via the intranasal route at six weeks of age, generated moderate immune responses and were poorly protective. However, neonatal DNA priming following by mucosal boosting with recombinant adenovirus generated strong immune responses, as evidenced by strong Ag85B-specific CD4+ and CD8+ T cell responses, both in the lung-associated lymph nodes and the spleen, by the quality of these responding cells (assessed by their capacity to secrete multiple antimicrobial factors), and by improved protection, as indicated by reduced bacterial burden in the lungs following pulmonary TB challenge. These results suggest that neonatal immunization with gene-based vaccines may create a favorable immunological environment that potentiates the pulmonary mucosal boosting effects of a subsequent heterologous vector vaccine encoding the same antigen. Our data indicate that immunization early in life with mycobacterial antigens in an appropriate vaccine setting can prime for protective immunity against Mtb.

Pneumocystis pneumonia is a major cause of morbidity and mortality in immunocompromised patients, particularly those infected with HIV. In this study, we evaluated the potential of oral immunization with live Pneumocystis to elicit protection against respiratory infection with Pneumocystis murina. C57BL/6 mice vaccinated with live P. murina using a prime-boost vaccination strategy were protected from a subsequent lung challenge with P. murina at 2, 7, 14, and 28 d postinfection even after CD4+ T cell depletion. Specifically, vaccinated immunocompetent mice had significantly faster clearance than unvaccinated immunocompetent mice and unvaccinated CD4-depleted mice remained persistently infected with P. murina. Vaccination also increased numbers of CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD11b+ macrophages in the lungs following respiratory infection. In addition, levels of lung, serum, and fecal P. murina–specific IgG and IgA were increased in vaccinated animals. Furthermore, administration of serum from vaccinated mice significantly reduced Pneumocystis lung burden in infected animals compared with control serum. We also found that the diversity of the intestinal microbial community was altered by oral immunization with P. murina. To our knowledge, our data demonstrate for the first time that an oral vaccination strategy prevents Pneumocystis infection.

Rationale Pneumocystis pneumonia is a major cause of morbidity and mortality in HIV-infected subjects, cancer patients undergoing chemotherapy and solid organ transplant recipients. No vaccine is currently available. By chemical labeling coupled with proteomic approach, we have identified a putative surface protein (SPD1, Broad Institute gene accession number PNEG_01848) derived from single suspended P. murina cysts. SPD1 was expressed in an insect cell line and tested for vaccine development. Methods Mice were immunized with SPD1 plus adjuvant MF-59 by subcutaneous injection. Three weeks after the last immunization, CD4+ cells were depleted with anti-CD4 antibody GK1.5. The mice were then challenged with 2 × 10 5 Pneumocystis organisms. Mice were sacrificed at 4 and 6 weeks after PC challenge. Spleen/lung cells and serum were harvested. B cells and memory B cells were assessed via flow cytometry. Specific Pneumocystis IgG antibody was measured by ELISA before and after challenge. Infection burden was measured as real-time PCR for P. murina rRNA. Results Normal mice infected with Pneumocystis mounted a serum IgG antibody response to SPD1. Serum from rhesus macaques exposed to Pneumocystis showed a similar serum IgG response to purified SPD1. SPD1 immunization increased B cell and memory B cell absolute cell counts in CD4-depleted Balb/c mice post Pneumocystis challenge in spleen and lung. Immunization with SPD1 significantly increased specific Pneumocystis IgG antibody production before and after challenge. Mice immunized with SPD1 showed significantly decreased P. murina copy number compared with mice that did not receive SPD1 at 6 weeks after challenge. Conclusion Immunization with SPD1 provides protective efficacy against P. murina infection. SPD1 protection against Pneumocystis challenge is associated with enhanced memory B cell production and higher anti– Pneumocystis IgG antibody production. SPD1 is a potential vaccine candidate to prevent or treat pulmonary infection with Pneumocystis .

Impairment of host immunity, particularly CD4+ T cell deficiency, presents significant complications for vaccine immunogenicity and efficacy. CD40 ligand (CD40L or CD154), a member of the tumor necrosis factor superfamily (TNFSF), is an important co-stimulatory molecule and, through interactions with its cognate receptor CD40, plays a pivotal role in the generation of host immune responses. Exploitation of CD40L and its receptor CD40 could provide a means to enhance and potentially restore protective immune responses in CD4+ T cell deficiency. To investigate the potential adjuvanticity of CD40L, we constructed recombinant plasmid DNA and adenoviral (Ad) vaccine vectors expressing murine CD40L and the mycobacterial protein antigen 85B (Ag85B). Co-immunization of mice with CD40L and Ag85B by intranasal or intramuscular prime-boosting led to route-dependent enhancement of the magnitude of vaccine-induced circulating and lung mucosal CD4+ and CD8+ T cell responses in both normal (CD4-replete) and CD4+ T cell deficient animals, including polyfunctional T cell responses. The presence of CD40L alone was insufficient to enhance or restore CD4+ T cell responses in CD4-ablated animals; however, in partially-depleted animals, co-immunization with Ag85B and CD40L was capable of eliciting enhanced T cell responses, similar to those observed in normal animals, when compared to those given vaccine antigen alone. In summary, these findings show that CD40L has the capacity to enhance the magnitude of vaccine-induced polyfunctional T cell responses in CD4+ T cell deficient mice, and warrants further study as an adjuvant for immunization against opportunistic pathogens in individuals with CD4+ T cell deficiency.

Background: Alcoholics are at heightened risk for developing active tuberculosis. This study evaluates chronic alcohol consumption in a murine model of vaccination with Mycobacterium bovis Bacille Calmette-Guerin (BCG) and subsequent pulmonary infection with virulent Mycobac- terium tuberculosis. Methods: BALB ⁄ c mice were administered the Lieber-DeCarli liquid ethanol diet or pair-fed the liquid control diet for 3 weeks either before or after subcutaneous vaccination with M. bovis BCG. At least 3 weeks after BCG vaccination, groups of mice on the aforesaid diets were chal- lenged with intratracheal infection with M. tuberculosis H37Rv. Lung mycobacterial burden, and lung and lung-associated lymph node CD4 + lymphocyte production of tuberculosis-specific interferon (IFN)-c were assayed. Popliteal lymph node lymphocytes from both dietary regimens undergoing BCG vaccination (in the absence of M. tuberculosis infection) were also evaluated for purified protein derivative-induced IFN-c production by ELISpot assay. Results: Mice begun on alcohol prior to vaccination with M. bovis BCG demonstrated impaired control of pulmonary challenge with virulent M. tuberculosis, as well as impaired lung CD4 + and popliteal lymph node T-cell IFN-c responses. If BCG vaccination was delivered prior to initiation of alcohol feeding, the mice remained protected against a subsequent challenge with M. tuberculosis, and BCG-induced immunity was not impaired in either the lung or the popliteal lymph nodes. Conclusions: Alcohol consumption blunts the development of the adaptive immune response to M. bovis BCG vaccination, which impairs the control of a secondary challenge with M. tuberculo- sis, but only if the alcohol exposure is begun prior to BCG vaccination. These results provide insight into mechanisms by which alcohol consumption impairs antimycobacterial immunity, including in response to vaccination and subsequent pathogenic challenge.

Significant safety issues have emerged concerning the general use of DRYVAX® vaccine. Vaccination with replication-defective recombinant adenovirus (rAd) vaccines may offer a safer and effective alternative to live vaccinia virus (VV) vaccination. Six individual poxvirus glycoproteins: A33R, A34R, A36R, B5R, A27L or L1R that are normally expressed on the surface of infectious vaccinia virus were encoded in rAd vaccines and tested in mice in this study. A single-shot intramuscular injection of rAd encoding A27L protected mice against a lethal intranasal challenge with VV at 4 weeks post vaccination. By 10 weeks post vaccination, a significant decrease in post-challenge morbidity was observed that correlated with potent neutralizing antibody responses and the emergence of specific polyfunctional T cell responses. The immunogenicity and protective efficacy of rAd-A27L immunization persisted for at least 35 weeks post-vaccination. This study is the first demonstration that a single-shot subunit vaccine encoding a poxvirus protein confers protection against the mortality and morbidity associated with poxvirus infection.

The type I IFNs exert a range of activities that include antiviral, antiproliferative, and immunomodulatory effects. To study this further, we have constructed recombinant vaccinia viruses expressing HIV or hemagglutinin (HA) Ags along with murine type I IFNs, IFN-alpha(4) (HA-VV-IFN-alpha(4)), IFN-beta (HA-VV-IFN-beta), or IFN-epsilon (HIV-VV-IFN-epsilon), a recently discovered member of this family. Our aims were to characterize IFN-epsilon functionality as a type I IFN and also to study the biological properties of these factors toward the development of safer and more effective vector-based vaccines. HIV-VV-IFN-epsilon and HA-VV-IFN-beta grew to lower titers than did their parental controls in murine cell lines. In vivo, however, HIV-VV-IFN-epsilon growth was not attenuated, while IFN-beta demonstrated potent local antiviral activity with no replication of HA-VV-IFN-beta detected. Flow cytofluorometric analysis of B lymphocytes incubated with virally encoded IFN-epsilon showed up-regulation of activation markers CD69 and CD86, while RT-PCR of IFN-epsilon-treated cells revealed that gene expression levels of antiviral proteins were elevated, indicating the induction of an antiviral state. The use of these constructs in a poxvirus prime-boost immunization regime led to robust humoral and cellular immune responses against the encoded Ags, despite the lack of replication in the case of HA-VV-IFN-beta. Thus, coexpression of these factors may be beneficial in the design of safer vector-based vaccines. Our data also indicate that while IFN-epsilon exhibits certain biological traits similar to other type I IFNs, it may also have a specific role in mucosal immune regulation that is quite distinct.

Flagellin has been tested as a protein-based vaccine adjuvant, with the majority of studies focused on antibody responses. Here, we evaluated the adjuvant activity of flagellin for both cellular and humoral immune responses in BALB/c mice in the setting of gene-based immunization, and have made several novel observations. DNA vaccines and adenovirus (Ad) vectors were engineered to encode mycobacterial protein Ag85B, with or without flagellin of Salmonella typhimurium (FliC). DNA-encoded flagellin given IM enhanced splenic CD4+ and CD8+ T cell responses to co-expressed vaccine antigen, including memory responses. Boosting either IM or intranasally with Ad vectors expressing Ag85B without flagellin led to durable enhancement of Ag85B-specific antibody and CD4+ and CD8+ T cell responses in both spleen and pulmonary tissues, correlating with significantly improved protection against challenge with pathogenic aerosolized M. tuberculosis. However, inclusion of flagellin in both DNA prime and Ad booster vaccines induced localized pulmonary inflammation and transient weight loss, with route-dependent effects on vaccine-induced T cell immunity. The latter included marked reductions in levels of mucosal CD4+ and CD8+ T cell responses following IM DNA/IN Ad mucosal prime-boosting, although antibody responses were not diminished. These findings indicate that flagellin has differential and route-dependent adjuvant activity when included as a component of systemic or mucosally-delivered gene-based prime-boost immunization. Clear adjuvant activity for both T and B cell responses was observed when flagellin was included in the DNA priming vaccine, but side effects occurred when given in an Ad boosting vector, particularly via the pulmonary route.

Background An important portion of asthmatics do not respond to current therapies. Thus, the need for new therapeutic drugs is urgent. We have demonstrated a critical role for PARP in experimental asthma. Olaparib, a PARP inhibitor, was recently introduced in clinical trials against cancer. The objective of the present study was to examine the efficacy of olaparib in blocking established allergic airway inflammation and hyperresponsiveness similar to those observed in human asthma in animal models of the disease. Methods We used ovalbumin (OVA)-based mouse models of asthma and primary CD4+ T cells. C57BL/6J WT or PARP-1−/− mice were subjected to OVA sensitization followed by a single or multiple challenges to aerosolized OVA or left unchallenged. WT mice were administered, i.p., 1 mg/kg, 5 or 10 mg/kg of olaparib or saline 30 min after each OVA challenge. Results Administration of olaparib in mice 30 min post-challenge promoted a robust reduction in airway eosinophilia, mucus production and hyperresponsiveness even after repeated challenges with ovalbumin. The protective effects of olaparib were linked to a suppression of Th2 cytokines eotaxin, IL-4, IL-5, IL-6, IL-13, and M-CSF, and ovalbumin-specific IgE with an increase in the Th1 cytokine IFN-γ. These traits were associated with a decrease in splenic CD4+ T cells and concomitant increase in T-regulatory cells. The aforementioned traits conferred by olaparib administration were consistent with those observed in OVA-challenged PARP-1−/− mice. Adoptive transfer of Th2-skewed OT-II-WT CD4+ T cells reversed the Th2 cytokines IL-4, IL-5, and IL-10, the chemokine GM-CSF, the Th1 cytokines IL-2 and IFN-γ, and ovalbumin-specific IgE production in ovalbumin-challenged PARP-1−/−mice suggesting a role for PARP-1 in CD4+ T but not B cells. In ex vivo studies, PARP inhibition by olaparib or PARP-1 gene knockout markedly reduced CD3/CD28-stimulated gata-3 and il4 expression in Th2-skewed CD4+ T cells while causing a moderate elevation in t-bet and ifn-γ expression in Th1-skewed CD4+ T cells. Conclusions Our findings show the potential of PARP inhibition as a viable therapeutic strategy and olaparib as a likely candidate to be tested in human asthma clinical trials.

Depletion or dysfunction of CD4+ T lymphocytes profoundly perturbs host defenses and impairs immunogenicity of vaccines. Here, we show that plasmid DNA vaccination with a cassette encoding antigen (OVA) and a second cassette encoding full-length CD40 ligand (CD40L), a molecule expressed on activated CD4+ T lymphocytes and critical for T cell helper function, can elicit significant titers of antigen-specific immunoglobulins in serum and Tc1 CD8+ T cell responses in CD4-deficient mice. To investigate whether this approach leads to CD4+ T cell-independent vaccine protection against a prototypic AIDS-defining infection, Pneumocystis (PC) pneumonia, we used serum from mice vaccinated with PC-pulsed, CD40L-modified DCs to immunoprecipitate PC antigens. Kexin, a PC antigen identified by this approach, was used in a similar DNA vaccine strategy with or without CD40L. CD4-deficient mice receiving DNA vaccines encoding Kexin and CD40L showed significantly higher anti-PC IgG titers as well as opsonic killing of PC compared with those vaccinated with Kexin alone. Moreover, CD4-depleted, Kexin-vaccinated mice showed a 3-log greater protection in a PC challenge model. Adoptive transfer of CD19+ cells or IgG to SCID mice conferred protection against PC challenge, indicating a role of humoral immunity in the protection. The results of these studies show promise for CD4-independent vaccination against HIV-related or other opportunistic pathogens.

Interleukin-10 (IL-10) is an anti-inflammatory and immune-deviating cytokine expressed in the endometrium and placenta. IL-10 null mutant (IL-10-/-) mice have been employed to examine the role of IL-10 in regulating immune events in early pregnancy and its significance in implantation and pregnancy success. The inflammatory response elicited in endometrial tissue by insemination was amplified in IL-10-/- mice, with a 66% increase in leukocytes in the endometrial stroma on Day 3 of pregnancy. Despite this, no evidence of abnormal type 1/type 2 skewing was seen in T-lymphocytes from lymph nodes draining the uterus. On Day 18 of gestation, IL-10-/- females mated with IL-10-/- males had 15% more implantation sites and 27% more viable fetuses than pregnant wild-type (IL-10+/+) mice. Placental weight was unaffected, but fetal weight and the fetal:placental weight ratio were higher in IL-10-/- pregnancies. Similar data were obtained in allogeneic pregnancies when IL-10-/- females were mated with major-histocompatibility complex (MHC) disparate IL-10-/- males. Pups delivered by IL-10-/- mothers had increased birth weight and followed an altered growth trajectory, with growth impairment evident from early postnatal life into adulthood, which was reflected in alterations in body composition at 14 wk of age. This study shows that although IL-10 is not essential for maternal immune tolerance or successful pregnancy irrespective of MHC disparity in the fetus, maternal IL-10 is a determinant of growth trajectory in progeny in utero and after birth.

Local production of IL-17 is a significant factor in effective host defense against Gram-negative bacteria. However, the proximal events mediating IL-17 elaboration by T cells remain unclear. In this study, we show in vivo that intact Toll-like receptor 4 signaling in the lung is required for induction of both the p19 transcript of IL-23 and IL-17 protein elaboration in response to Klebsiella pneumoniae. Although IL-17 is widely considered a CD4+ T cell product, we also demonstrate significant in vitro IL-17 production by CD8+ T cells after culture in medium from dendritic cells exposed to these bacteria. The dominant portion of this IL-17-inducing activity for both CD4+ and CD8+ T cells is IL-23. These data demonstrate the critical signaling pathway for IL-17 induction in the host response to Gram-negative pulmonary infection and suggest a direct role for IL-23 in CD8+ T cell IL-17 production.

Murine interferon-inducible T cell alpha chemokine (I-TAC) is a potent non-ELR Cys-X-Cys (CXC) chemokine that predominantly attracts activated T lymphocytes and binds to the receptor CXCR3. Using semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) we analysed murine I-TAC expression in two different progenitor dendritic cell (DC) lines, MTHC-D2 and JAWS II which were exposed to various cytokines, and Con A-activated splenocytes from a panel of knockout mice. Analysis of the progenitor DC lines and Con A cultures demonstrated that murine I-TAC is primarily regulated by interferon (IFN)-γ via interferon regulatory factor (IRF)-1. It has been proposed that I-TAC may have a role in autoimmune diseases such as multiple sclerosis (MS). Because I-TAC appears to be secreted from antigen-presenting cells (APCs) and attracts activated T cells, we examined the level of murine I-TAC mRNA in the central nervous system (CNS) of wild-type and IFN-γ-receptor knockout (IFN-γR−/−) mice with myelin oligodendrocyte glycoprotein (MOG)35–55 peptide-induced experimental autoimmune encephalomyelitis (EAE). Peak I-TAC expression was detected in wild-type mice on day 14 when the mice begin to recover, whereas very low levels of I-TAC were detected in the CNS of IFN-γR−/− mice which develop severe EAE and die. The expression characteristics of murine I-TAC suggest an important mediator of immune cell communication that could augment vaccines and autoimmune therapies.

Mice immunised with oxidised mannan-MUC1 fusion protein (M-FP) develop MHC restricted CD8(+) cytotoxic T cells. We now demonstrate that in MUC1/HLA-A2 transgenic mice, IL-12 gives enhanced CTL, CTLp and tumor protection. CTLp in MUC1 transgenic mice with M-FP were 1/55,000, and with IL-12, this increased to 1/19,000, with improved tumor protection. Thus, IL-12 is important for effective CTL responses to MUC1 in transgenic mice.

The mechanisms regulating the selective migration and degranulation of eosinophils in the asthmatic lung and the subsequent development of airways hyperreactivity (AHR) have not been fully delineated. In this investigation, we have employed a novel transgene model to facilitate the dissection of the contributions of IL-5 and/or eotaxin to eosinophil function in the absence of complex tissue signals derived from the allergic lung. Gene transfer of IL-5 and/or eotaxin to the lungs of naive mice induced a pronounced and selective airways eosinophilia, but did not result in eosinophil degranulation or AHR. Airways eosinophilia occurred independently of the induction of a blood eosinophilia, but was markedly augmented by the coexpression of both cytokines and/or by the transient mobilization of eosinophils from the bone marrow by the administration of i.v. IL-5. However, for eosinophil degranulation and AHR to occur, the inhalation of Ag was required in association with IL-5 and eotaxin expression. Investigations in IL-5-deficient mice linked eosinophilia, and not solely IL-5 and eotaxin, with the induction of AHR. Furthermore, eosinophil degranulation and AHR were dependent on CD4+ T cells. Importantly, this investigation shows that IL-5 regulates eosinophilia within the lung as well as in the circulation and also amplifies eotaxin-induced chemotaxis in the airway compartment. Moreover, the interplay between these cytokines, CD4+ T cells, and factors generated by Ag inhalation provides fundamental signals for eosinophil degranulation and the induction of AHR.

In this article, we describe several novel genetic vaccination strategies designed to facilitate the development of different types of immune responses. These include: i) the consecutive use of DNA and fowlpoxvirus vectors in "prime-boost" strategies which induce greatly enhanced and sustained levels of both cell-mediated immunity and humoral immunity, including mucosal responses; ii) the co-expression of genes encoding cytokines and cell-surface receptors, and the use of immunogenic carrier molecules, for immune modulation and/or improved targeting of vector-expressed vaccine antigens; and iii) the expression of minimal immunogenic amino acid sequences, particularly cytotoxic CD8+ T-cell determinants, in "polytope" vector vaccines. The capacity to modulate and enhance specific immune responses by the use of approaches such as these may underpin the development of vaccines against diseases for which no effective strategies are currently available.

Allergic asthma strongly correlates with airways inflammation driven by interleukin (IL)-4 and IL-5 secreted by allergen-specific CD4+ T cells. It is possible that over-production of these factors in the lungs may render asthmatic individuals less able to resolve virus infection of the respiratory tract by down-regulating type 1 cytokine-driven immune responses. IL-12 is important for the establishment of cell-mediated immunity (CMI) and may also inhibit responses driven by type 2 cytokine production. Sustained expression of IL-12 in the airways may, therefore, represent an effective preventive treatment or therapy for allergic asthma and any adverse consequences of excessive production of type 2 cytokines for the development of local CMI. Here, we show that allergic responses in airways profoundly inhibit the development of antiviral CMI in mice following local immunization with vaccinia virus (VV) leading to persistent lung infection. However, mucosal gene transfer of IL-12 in the lung, via a VV vector, inhibited local type 2 cytokine production, both prevented the development of allergic disease and airways hyperreactivity in a manner largely dependent on endogenous interferon-gamma expression and suppressed established allergic disease, and reversed the suppression of local antiviral CMI responses resulting in rapid resolution of virus infection. Our study provides the first direct demonstration that allergic conditions, particularly in airways, may inhibit immune responses to concomitant virus infection and suggests that transient mucosal IL-12 gene therapy represents an effective approach to both the prevention and treatment of allergic airways disease and associated immunosuppression of CMI.

We have engineered recombinant vaccinia virus vectors expressing murine interleukin-7 (IL-7) in order to study the activity of this factor during virus infection. Virus-encoded IL-7 dramatically increased splenic cellularity in infected mice and enhanced the proliferative activity of T cells and their capacity to secrete IL-2 and IL-6, but not IFN-gamma, TNF-alpha or IL-4. Numbers of splenic CD4+ and CD8+ T lymphocytes were elevated two- to threefold. IL-7 also mediated a marked enhancement of both antigen-specific and nonspecific cellular immune activity. Total splenic antiviral cytotoxic T cells (CTL), natural killer (NK), and lymphokine-activated killer cells (LAK) responses were augmented significantly in mice given VV-HA-IL-7 compared with those given control virus, with accelerated clearance of the former. The enhanced antiviral cellular immune activity mediated by IL-7 was critically dependent on IL-2 produced by the host, but occurred independently of IFN-gamma. The ability of IL-7 to induce cellular immune responses in vivo may have applications in antiviral immunotherapy, particularly in cases of immunodeficiency.

We have shown previously both in vitro and in vivo that IL-6 is an important factor for the development of IgA-producing B cells. However, despite the lack of this cytokine in mice with targeted disruption of the interleukin (IL)-6 gene (gene knockout mice), a substantial number of IgA-producing plasma cells occur in their intestinal mucosa. The experiments reported here indicate that there is a population of IgA-producing B cell precursors originating from the peritoneal cavity, distinguished from conventional Peyer's patch-derived precursors by their expression of CD5, and that IgA secretion by these cells is IL-6-independent. Further, there is an increase in CD5 expression among brightly staining IgA-producing cells obtained from the intestinal lamina propria of IL-6 gene-disrupted mice compared to normal controls. These data suggest an explanation for the persistence of IgA-producing plasma cells in the intestinal mucosa of IL-6-depleted mice and indicate the importance of IL-6 for development of conventional precursors of IgA-producing B cells, but not those derived from the peritoneal cavity pool.

Avipoxviruses have recently been studied as potential vectors for the delivery of heterologous vaccine antigen. Because these viruses abortively infect mammalian cells yet still effectively present encoded foreign genes to the host immune system, they offer a safer but effective alternative to other live virus vectors. We have examined the effect of coexpressing the cytokine interleukin-6 or gamma interferon on immune responses to a recombinant fowlpox virus expressing influenza virus hemagglutinin. The encoded cytokine was expressed for prolonged periods in infected cell culture with little cytopathic effect due to the abortive nature of the infection. In mice, vector-expressed cytokine dramatically altered immune responses induced by the coexpressed hemagglutinin antigen. Expression of interleukin-6 augmented both primary systemic and mucosal antibody responses and primed for enhanced recall responses. In contrast, expression of gamma interferon markedly inhibited antibody responses without affecting the generation of cell-mediated immunity. The safety of these constructs was demonstrated in mice with severe combined immunodeficiency, and no side effects due to cytokine expression were observed. In summary, fowlpox virus vectors encoding cytokines represent a safe and effective vaccine strategy which may be used to selectively manipulate the immune response.

We have examined the potential of a novel cytokine, interleukin-27 (IL-27), for gene therapy of prostate cancer. IL-27 is the most recently characterized member of the family of heterodimeric IL-12-related cytokines and has shown promise in halting tumor growth and mediating tumor regression in several cancer models. In the present study, we examined the efficacy of a new mode of gene delivery to prostate tumors: low-frequency ultrasound irradiation or "sonoporation." We also examined the potential of IL-27 gene delivery by sonoporation to treat and reduce the growth of prostate cancer in vivo. We used three models of immune-competent prostate adenocarcinoma and characterized the tumor-growth reduction, gene-profile expression, and effector cellular profiles. Our results suggest that IL-27 can be effective in reducing tumor growth and can help enhance accumulation of effector cells in prostate tumors in vivo. These results are promising, because they are potentially relevant to developing novel therapies that can be translated by using the novel and effective sonoporation gene-therapy delivery strategy.

Generation and isolation of recombinant herpesviruses by traditional homologous recombination methods can be a tedious, time-consuming process. Therefore, a novel stoplight recombination selection method was developed that facilitated rapid identification and purification of recombinant viruses expressing fusions of immunological epitopes with EGFP. This “traffic-light” approach provided a visual indication of the presence and purity of recombinant HSV-1 isolates by producing three identifying signals: (1) red fluorescence indicates non-recombinant viruses that should be avoided; (2) yellow fluorescence indicates cells co-infected with non-recombinant and recombinant viruses that are chosen with caution; (3) green fluorescence indicates pure recombinant isolates and to proceed with preparation of viral stocks. Adaptability of this system was demonstrated by creating three recombinant viruses that expressed model immunological epitopes. Diagnostic PCR established that the fluorescent stoplight indicators were effective at differentiating between the presence of background virus contamination and pure recombinant viruses specifying immunological epitopes. This enabled isolation of pure recombinant viral stocks that exhibited wildtype-like viral replication and cell-to-cell spread following three rounds of plaque purification. Expression of specific immunological epitopes was confirmed by western analysis, and the utility of these viruses for examining host immune responses to HSV-1 was determined by a functional T cell assay.

In this study we have firstly compared a range of recombinant DNA poxvirus prime-boost immunisation strategies and shown that combined intramuscular (i.m.) 2× DNA-HIV/intranasal (i.n.) 2× FPV-HIV prime-boost immunisation can generate high-level of HIV-specific systemic (spleen) and mucosal (genito-rectal nodes, vaginal tissues and lung tissues) T cell responses and HIV-1 p24 Gag-specific serum IgG1, IgG2a and mucosal IgG, SIgA responses in vaginal secretions in BALB/c mice. Data indicate that following rDNA priming, two rFPV booster immunisations were necessary to generate good antibody and mucosal T cell immunity. This data also revealed that mucosal uptake of recombinant fowl pox (rFPV) was far superior to plasmid DNA. To further evaluate CD8+ T cell immunity, i.m. 2× DNA-HIV/i.n. 1× FPV-HIV immunisation strategy was directly compared with single shot poxvirus/poxvirus, i.n. FPV-HIV/i.m. VV-HIV immunisation. Results indicate that the latter strategy was able to generate strong sustained HIV-specific CD8+ T cells with higher avidity, broader cytokine/chemokine profiles and better protection following influenza-K d Gag 197–205 challenge compared to rDNA poxvirus prime-boost strategy. Our findings further substantiate the importance of vector selection/combination, order and route of delivery when designing effective vaccines for HIV-1.

Several in vitro studies have shown that murine interleukin-5 (mIL-5) enhances IgA production by activated mucosal B cells. To date, however, there is no evidence that this factor significantly up-regulates mucosal IgA responses in vivo. Here, we show that expression of the gene for mIL-5 in a recombinant vaccinia virus vector markedly increases IgA responses to co-expressed heterologous antigen in the lungs of mice given intranasal inocula of the virus. The elevated local IgA responses to vectors expressing mIL-5 peaked at a fourfold higher level than those elicited by control virus at 14 days after infection and were sustained for at least 4 weeks. Increased IgA responses were abrogated in mice treated with monoclonal antibody against mIL-5 and were not detected in systemic lymphoid tissue. No enhancement of specific IgG levels was found either locally or systemically. Our results indicate that mIL-5 selectively enhances the development of mucosal IgA responses in vivo and suggest that expression of this factor in mucosal vaccine vectors may stimulate local immune reactivity.

Objective Interleukin (IL)-17, which now defines the Th 17 immune response, is a critical cytokine expressed and required for stress granulopoiesis during microbial invasion. Dendritic cells (DC) can instigate this response by inducing IL-17 expression in CD4 + T cells. Besides IL-17, microbial invasion also stimulates production of the DC growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). The objective was the in vitro and in vivo investigation of IL-17 on DC proliferation and function in mice. Materials and Methods Murine IL-17 (mIL-7) or murine GM-CSF (mGM-CSF), or both, was expressed in C57BL6 mice using adenoviral technology to assess hematopoietic and DC changes. The E-22 tymoma tumor cell line using a previously described vaccinia virus ovalbumin/LacZ murine tumor model was employed to study effects on tumor rejection. Results The combination of mIL-17 and mGM-CSF increased peripheral neutrophila by 28-fold and splenic colonies by 11- and 14-fold over each individual factor in mice, respectively. The effect of mIL-17 by itself on murine DCs in vitro and in vivo was minimal; however, the combination greatly enhanced the stimulating effects of mGM-CSF, increasing the total numbers of CD14b/c + spleen DC by fourfold, as well as their function measured by enhanced endocytosis. Mixed lymphocyte reactions using mIL-17/mGM-CSF cultured DCs stimulator cells enhanced lymphocyte responses by twofold over mGM-CSF alone. Vaccination against LacZ in the C57BL6 E22 syngenic thymoma tumor model effectively delayed tumor growth in animals pretreated with the mIL-17/mGM-CSF combination prior to vaccination. Conclusions mIL-17 effectively synergizes with mGM-CSF in stimulating granulopoiesis and DC expansion, as well as in functional enhancement of DCs.

Athymic, nude mice, which normally succumb to virus infection, can resolve infection with recombinant vaccinia virus (rVV) engineered to express IL-2. We have demonstrated that interferon-gamma (IFN-gamma) produced by natural killer (NK) cells and other immunocytes in response to the virus-encoded interleukin-2 (IL-2) is crucial to recovery. Here, we extend this work to show that nude mice, when primed intravenously with rVV co-expressing both IL-2 and an influenza virus haemagglutinin (HA) gene, are also protected following challenge with a lethal dose of homologous influenza virus. A substantial increase in the number of influenza virus-reactive antibody-secreting cells producing antibody of the IgM isotype, but not of the IgG or IgA isotypes, was found in spleens and lungs of the protected mice. Treatment with monoclonal antibodies to IFN-gamma or to the NK marker, as GM1, at challenge and thereafter, led to their death however, though the specific IgM antibody response was unaffected. These data suggest that both specific antibody and non-specific antiviral reactivity are important elements of the protective response and show that this immunization strategy may be used to protect severely immunocompromised individuals.

Unprecedented levels of cell-mediated immunity can be stimulated by the consecutive use of DNA vaccines and attenuated poxvirus vectors encoding similar heterologous antigens. This may offer a means of preventive vaccination against diseases for which no effective treatment is available.

There is an urgent need for effective prophylactic measures against Mycobacterium tuberculosis (Mtb) infection, particularly given the highly variable efficacy of Bacille Calmette-Guerin (BCG), the only licensed vaccine against tuberculosis (TB). Most studies indicate that cell-mediated immune responses involving both CD4+ and CD8+ T cells are necessary for effective immunity against Mtb. Genetic vaccination induces humoral and cellular immune responses, including CD4+ and CD8+ T-cell responses, against a variety of bacterial, viral, parasitic and tumor antigens, and this strategy may therefore hold promise for the development of more effective TB vaccines. Novel formulations and delivery strategies to improve the immunogenicity of DNA-based vaccines have recently been evaluated, and have shown varying degrees of success. In the present study, we evaluated DNA-launched Venezuelan equine encephalitis replicons (Vrep) encoding a novel fusion of the mycobacterial antigens α-crystallin (Acr) and antigen 85B (Ag85B), termed Vrep-Acr/Ag85B, for their immunogenicity and protective efficacy in a murine model of pulmonary TB. Vrep-Acr/Ag85B generated antigen-specific CD4+ and CD8+ T cell responses that persisted for at least 10 wk post-immunization. Interestingly, parenterally administered Vrep-Acr/Ag85B also induced T cell responses in the lung tissues, the primary site of infection, and inhibited bacterial growth in both the lungs and spleens following aerosol challenge with Mtb. DNA-launched Vrep may, therefore, represent an effective approach to the development of gene-based vaccines against TB, particularly as components of heterologous prime-boost strategies or as BCG boosters.

We have tested the efficacy of recombinant fowl pox (rFPV) and recombinant vaccinia virus (rVV) encoding antigens of AE clade HIV-1 in a prime-boost strategy, using both systemic and mucosal delivery routes. Of the various vaccine routes tested, intranasal/intramuscular (i.n./i.m.) AE FPV/AE VV prime-boosting generated the highest mucosal and systemic T cell responses. Peak mucosal T cell responses occurred as early as 3 days post-boost vaccination. In contrast only low systemic responses were observed at this time with the peak response occurring at day 7. Current data also revealed that, due to better uptake of the rFPV, intranasal viral priming was much more effective than intranasal rDNA priming tested previously. The i.m./i.m. prime-boost delivery also generated strong systemic but poor mucosal responses to Gag peptides. Interestingly, the oral administration of AE FPV followed by i.m. AE VV delivery elicited strong systemic responses to sub-dominant Pol 1 peptides that were absent in mice that received vaccine by other routes. Moreover, priming with AE FPV co-expressing cytokine IL-12 significantly enhanced the T cell responses to target antigens, whilst co-expression of IFNgamma decreased these responses. The results also indicated that the route of inoculation and the vaccine vector combination could radically influence not only the magnitude but also the antigen specificity of the immune response generated. Further, in contrast to the generally protracted HIV rDNA/rFPV multiple delivery prime-boosting, this single rFPV prime and rVV boost approach was more flexible and generated excellent mucosal and systemic immune responses to HIV vaccine antigens.

Interleukin-23 (IL-23) is a heterodimeric cytokine that shares IL-12 p40 but contains a unique p19 subunit similar to IL-12 p35. Previous studies indicate a greater importance for intact IL-12/23 p40 expression than IL-12 p35 for immunity against Mycobacterium tuberculosis , suggesting a role for IL-23 in host defense. The effects of IL-23 on the outcome of pulmonary infection with M. tuberculosis have not been described. Here, we show that local delivery of replication-defective adenovirus vectors encoding IL-23 (AdIL-23) greatly stimulated expression of both gamma interferon (IFN-γ) and IL-17 in lung tissues of otherwise normal mice. When given 72 h prior to infection with M. tuberculosis , AdIL-23 significantly reduced the bacterial burden at 14, 21, and 28 days. Markedly lower levels of lung inflammation were observed at 28 days than in control mice pretreated with control adenovirus (AdNull) or vehicle controls. AdIL-23 pretreatment resulted in increased numbers of CD4 + CD25 + activated T cells in lungs and draining lymph nodes compared to control groups and more CD4 + T cells bearing surface memory markers in lung lymph nodes. IL-23 gene delivery also significantly enhanced host anti-mycobacterial T-cell responses, as shown by elevated levels of IFN-γ and IL-17 secreted in vitro following restimulation with M. tuberculosis purified protein derivative. Overall, our data show that transient IL-23 gene delivery in the lung is well tolerated, and they provide the initial demonstration that this factor controls mycobacterial growth while augmenting early pulmonary T-cell immunity.

Further advances are required in understanding protection from AIDS by T cell immunity across mucosal sites of virus transmission. We analysed a set of multigenic HIV and SHIV DNA and Fowlpoxvirus (FPV) prime and boost vaccines for immunogenicity and protective efficacy in outbred pigtail macaques when delivered via mucosal surfaces (intranasally or intrarectally). Intranasally delivered DNA, even when adjuvanted and given as a fine droplet spray, was neither immunogenic nor protective in macaques. Some protection from acute infection with a pathogenic vaginal SHIVSF162P3 challenge was, however, observed with a regimen involving intramuscular DNA vaccine priming followed by either intranasally or intrarectally delivered rFPV boosting. Interestingly, animals boosted with rFPV vaccine via either of these mucosal routes had poor circulating T cell responses prior to challenge with SHIV compared to those boosted via the intramuscular route. Nevertheless, the mucosally-vaccinated animals generated equivalent anamnestic mucosal and systemic SHIV-specific CD4 and CD8 T cell responses following SHIV administration, with significant reduction in acute plasma viremia against this vaginal challenge. Our data suggest strategies for effective priming of partial immunity to mucosal HIV-1 exposure utilizing systemic prime and mucosal boost vaccination strategies.

Cytotoxic T lymphocytes (CTLs) are crucial effectors against intracellular pathogens and cancer. Accurate and efficient assessment of CTL activity is important for basic and clinical studies. Widely used CTL assays, including the chromium release, JAM test and ELISPOT, involve either radioisotopes or lengthy procedures. Here, we developed a new fluorolysometric CTL assay based on cell-mediated cytolysis of fluorescent protein (GFP or DsRed) expressing cells quantified by one of the fluoro-based methods: flow cytometry, fluorescence microplate reader, or fluorescence microscopy. With flexible detection methods and lentiviral vector transduced stable lines of either GFP+ or DsRed+ cells as targets for antigen presentation and equal number of the other as internal reference for consistency and accuracy, this assay is easy to perform and to scale-up for simultaneous multi-sample analyses. Using two different antigen systems, we demonstrated that this assay is very sensitive to determine primary CTL activity of both in vitro and in vivo primed antigen-specific T cells. Thus, this FL-CTL assay is highly sensitive, reliable, reproducible, economical, convenience and supports broad applications compared to conventional CTL assays.

Induction of HIV-specific T-cell responses by vaccines may facilitate efficient control of HIV. Plasmid DNA vaccines and recombinant fowlpoxvirus (rFPV) vaccines are promising HIV-1 vaccine candidates, although either vaccine alone may be insufficient to protect against HIV-1. A consecutive immunisation strategy involving priming with DNA and boosting with rFPV vaccines encoding multiple common HIV-1 antigens was further evaluated in 30 macaques. The DNA vaccine vector included CpG immunostimulatory molecules, and rFPV vaccines were compared with rFPV vaccines co-expressing the pro-T cell cytokines IFNgamma or IL-12. Vaccines expressed multiple HIV-1 genes, mutated to remove active sites of the HIV proteins. The vaccines were well tolerated, and a significant enhancement of DNA-vaccine primed HIV-1 specific T lymphocyte responses was observed following rFPV boosting. Co-expression of IFNgamma or IL-12 by the rFPV vaccines did not further enhance immune responses. Non-sterilising protection from a non-pathogenic HIV-1 challenge was observed. This study provides evidence of a safe, optimised, strategy for the generation of T-cell mediated immunity to HIV-1.

Improved vaccines and therapies for virulent poxvirus infection are required, particularly in the light of recent threats of bioterrorism. Cidofovir (HPMPC) is an acyclic nucleoside analog with proven efficacy against poxviruses. Here, we evaluated HPMPC in mice given a recombinant ectromelia virus (ECTV) encoding interleukin-4 (ECTV-IL-4) that is highly immune suppressive. Mousepox-sensitive BALB/c mice given HPMPC for five consecutive days after infection were protected against the lethal effects of a control ECTV recombinant, although they suffered a chronic form of mousepox disease. High doses of the drug resulted in a milder localized disease. In contrast, HPMPC failed to protect mousepox-resistant C57BL/6 mice against ECTV-IL-4, although its lethal effects were delayed by five daily doses of 20 mg/kg or a single dose of 100 mg/kg. Higher daily doses further delayed mortality, although the majority of animals eventually succumbed to infection. It appears that HPMPC inhibited ECTV-IL-4 replication without clearance, with the virus having a lethal effect when the drug was removed. Resistance of ECTV-IL-4 to HPMPC treatment may relate to the virus's ability to inhibit antiviral cell-mediated immunity. Interestingly, ECTV-IL-4-mediated immune suppression was not accompanied by a reduction in systemic IFN-gamma expression, suggestive of an alternative or highly localized suppressive mechanism.

Placental morphogenesis and nutrient transfer function are regulated by growth factors at the foeto-maternal interface. Interleukin (IL)-10, expressed in the decidua and placenta, is implicated in regulating extravillous cytotrophoblast invasion through inhibiting matrix metalloproteinase expression and influencing the quality of the maternal immune response. Our laboratory has previously found that IL-10 deficiency in both the mother and foetus increases foetal weight at day 18 without altering placental weight suggesting that the placenta has a greater functional capacity when IL-10 is absent. The present study has used IL-10 null mutant (IL-10−/−) mice to investigate the role of IL-10 in placental development. Placental structure was assessed in adult virgin IL-10−/− or wild-type (IL-10+/+) mice mated with males of the same genotype and sacrificed at day 18 of gestation. Mid-sagittal cross sections of placental tissue were stained with Masson's trichrome or immuno-labelled with MTS-12 and pan-cytokeratin reactive antibodies to identify foetal endothelial cells and trophoblasts, respectively, and examined with video image analysis. IL-10 deficiency increased the total cross sectional area of the placenta by 28 per cent (IL-10−/− n =22 placentae from 8 dams, IL-10+/+ n =21 placentae from 9 dams, P =0.026), principally through increasing the cross sectional area of placental labyrinth by 37 per cent ( P =0.025). The proportion of maternal blood space in the labyrinth was increased by 26 per cent ( P =0.001) and that of trophoblast was decreased by 16 per cent ( P =0.001) in IL-10−/− placentae. The surface area of trophoblast per gram of labyrinth was increased by 41 per cent ( P =0.0005) in IL-10−/− placentae. In the absence of IL-10, structural correlates of placental function are enhanced consistent with concomitant increases in foetal growth. These data indicate that IL-10 is a regulator of placental morphogenesis, acting to retard expansion of the placental labyrinth and to modify the architecture of the maternal blood sinuses. Since previous studies have paradoxically shown that postnatal growth is impaired in IL-10 null mutants, these observations are consistent with a role for IL-10 in regulating placental development and foetal programming.

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.

Some cytokines are known to have potent antiviral activity in vitro, and recent work shows that severely immunodeficient mice, which lack conventional effector T cells, can still recover from virus infection provided these factors are present at sites of virus replication. Here Alistair Ramsay, Janet Ruby and Ian Ramshaw discuss these findings and raise fundamental questions concerning the physiological role of cytotoxic T cells in the resolution of virus infection.

Development and expansion of high-avidity T cell populations may be important for the success of immunization strategies against HIV and other pathogens that have presented major problems for vaccine development. We have used tetrameric-MHC complexes ex vivo and lytic assays to show that 'prime-boost' immunization with DNA vaccines and recombinant poxvirus vectors generates high frequencies of cytotoxic T lymphocytes (CTL) that recognize target cells expressing very low levels of specific antigen. These cells persist for at least 6 months at levels representing approximately 10% of the CD8(+) T cell population. Using a novel in vivo assay, we also found that prime-boost immunized animals were capable of eliminating target cells expressing 10- to 100-fold less immunogenic peptide than mice given either vector alone. In addition, viral challenge led to rapid expansion of CTL effectors in prime-boost groups, to levels representing30% of total CD8(+) T cell numbers. Strategies that generate specific T cells of high avidity, optimizing early detection of infected cells, offer new hope for effective prophylaxis and immunotherapy.

Genetic resistance to clinical mousepox (ectromelia virus) varies among inbred laboratory mice and is characterized by an effective natural killer (NK) response and the early onset of a strong CD8 1 cytotoxic T-lymphocyte (CTL) response in resistant mice. We have investigated the influence of virus-expressed mouse interleukin-4 (IL-4) on the cell-mediated response during infection. It was observed that expression of IL-4 by a thymidine kinase-positive ectromelia virus suppressed cytolytic responses of NK and CTL and the expression of gamma interferon by the latter. Genetically resistant mice infected with the IL-4-expressing virus developed symptoms of acute mousepox accompanied by high mortality, similar to the disease seen when genetically sensitive mice are infected with the virulent Moscow strain. Strikingly, infection of recently immunized genetically resistant mice with the virus expressing IL-4 also resulted in significant mortality due to fulminant mousepox. These data therefore suggest that virus-encoded IL-4 not only suppresses primary antiviral cellmediated immune responses but also can inhibit the expression of immune memory responses. Ectromelia virus (ECTV; family Poxviridae, genus Orthopoxvirus) is a natural pathogen of laboratory mice that causes a generalized disease termed mousepox (13). All mice are equally susceptible to infection by footpad inoculation; however, development of clinical mousepox among inbred mouse strains differs greatly (44). In mousepox-sensitive (e.g., BALB/c) mice, the disease is an acute systemic infection with high viral titers in the liver and spleen with resultant necrosis and high mortality. In contrast, infection of mousepox-resistant (e.g., C57BL/6) mice is usually subclinical, with lower levels of viral replication in the visceral organs and development of nonfatal lesions. Genetic resistance has been found to act through the combined activity of innate host defenses including natural killer (NK) cells, alpha interferon (IFN-a), IFN-b, IFN-g, ac

Although the chain of events leading to asthma is highly complex, the dysregulated production of type 2 cytokines, notably IL-4 and IL-5, by allergen-specific T cells, has emerged as an important causative factor [1, 2]. The symptoms of allergic asthma, including reversible airways occlusion and airways hyperreactivity (AHR), correlate with a local infiltration of the airways mucosa by inflammatory cells, whose recruitment and activation is influenced by IL-3, IL-4, IL-5, granulocyte-macrophage colony stimulating factor and other chemotactic agents produced by activated Th2 cells [3-6]. Indeed, the severity of disease appears to correlate with the degree of airways inflammation and the level of local Th2 cell activity [7, 8]. The molecular and cellular mechanisms underlying the presence of large numbers of Th2 cells in allergic airways remain to be resolved, however their products appear to have profound and specific effects in promoting allergic disease. IL-5, for example, regulates all stages of the development of eosinophils, which are consistently the predominant leukocyte population in the inflammatory infiltrate of asthmatics [5]. IL-4 modulates mast cell activity [4] and, through its influence on Ig class switching, regulates the production of IgE [3] which may specifically sensitize inflammatory cells implicated in allergic airways disease, including mast cells, eosinophils, basophils and macrophages. This leads to the release of inflammatory substances including histamine, leukotrienes, platelet-activating factor, cationic proteins and chemokines which may, in turn, augment the inflammatory process.

The feasibility of using viral constructs expressing cytokine genes to influence the course of bacterial infection was tested in mice. The mice were first infected with vaccinia or fowlpox viruses expressing the cytokine of interest, then challenged with the facultative intracellular bacterial pathogen Listeria monocytogenes. The course of infection was assessed by subsequent bacterial counts. Expression of IFN-gamma or TNF was protective. Vaccinia virus was more efficient at delivering IFN-gamma-mediated protection than was fowlpox virus, which is unable to proliferate in mammalian cells. The effect of vaccinia-IFN-gamma was more apparent in the liver, where vaccinia proliferates to high titres (109), than in the spleen, where only 103 vaccinia were isolated. Vaccinia virus expressing IL-4 exacerbated infection. Interleukin-4 exacerbation was T cell independent and was reflected in the failure of macrophage activation, possibly due to suppression of NK cells, which are a source of IFN-gamma early in infection. The clear indication of protection by some cytokines in this prophylactic model appears to justify further study of the therapeutic effects of cytokine-expressing viruses in chronic bacterial infections, especially where a cytokine defect is suspected.

During the past year, significant advances have been made in our understanding of cytokine regulation and the respective roles played by T helper cells type 1 and 2 immune responses during virus infection. Numerous mechanisms by which viruses may evade host immune defences have now been identified, some directly influencing cytokine activity. Major advances have also been made in delineating the roles of cytokines and chemokines at different stages in the pathogenesis of HIV infection.

In this review, we discuss two broad approaches we have taken to study the role of cytokines and chemokines in antiviral immunity. Firstly, recombinant vaccinia viruses were engineered to express genes encoding cytokines and chemokines of interest. Potent antiviral activity was mediated by many of these encoded factors, including IL-2, IL-12, IFN-gamma, TNF-alpha, CD40L, Mig and Crg-2. In some cases, host defense mechanisms were induced (IL-2, IL-12, Mig and Crg-2), whilst for others, a direct antiviral effect was demonstrated (IFN-gamma, TNF-alpha and CD40L). In sharp contrast, vector-directed expression of IL-4, a type 2 factor, greatly increased virus virulence, due to a downregulation of host type 1 immune responses. Our second experimental approach involved the use of strains of mice deficient for the production of particular cytokines or their receptors, often in combination with our engineered viruses. Mice deficient in either IFN-gamma, IFN-gamma R, IFN-alpha/beta R, TNFRs, CD40 or IL-6 were, in general, highly susceptible to poxvirus infection. Surprisingly, not only the TNFR1, but also the TNFR2, was able to mediate the antiviral effects of TNF-alpha in vivo, whilst the antiviral activity observed following CD40-CD40L interaction is a newly defined function which may involve apoptosis of infected cells. Through the use of perforin-deficient mice, we were able to demonstrate a requirement for this molecule in the clearance of some viruses, such as ectromelia virus, whilst for others, such as vaccinia virus, perforin was less important but IFN-gamma was essential.

Recent demonstrations of the immunogenicity of antigens encoded in DNA plasmids following delivery by various routes have heralded a new era in vaccine development. In this article, we review progress in DNA-based antiviral immunoprophylaxis. Preclinical studies have already established the immunogenicity of DNA plasmids encoding protective antigens from a wide variety of viral pathogens and work published in recent months has raised real prospects of broadly protective DNA vaccination against infections with influenza virus and HIV. We also describe a consecutive immunization protocol consisting of a priming dose of vaccine antigen encoded in DNA plasmids followed by a booster with the same antigen encoded in recombinant fowlpox virus vectors. We have used this strategy to generate protective antiviral cell-mediated immunity and sustained, high-level antibody responses both systemically and at mucosae, and to elucidate immunological mechanisms underlying the development of immunity to antigens delivered in DNA vectors.

In this investigation we have used a mouse model containing certain phenotypic characteristics consistent with asthma and IL-4- and CD40-deficient mice to establish the role of this cytokine and allergen-specific immunoglobulins in the initiation of airways hyperreactivity and morphological changes to the airways in responses to aeroallergen challenge. Sensitization and aerosol challenge of mice with ovalbumin resulted in a severe airways inflammatory response which directly correlated with the induction of extensive airways damage and airways hyperreactivity to beta-methacholine. Inflammatory infiltrates were primarily characterized by the presence of CD4+ T cells and eosinophils. In IL-4-deficient mice, the recruitment of airways eosinophils was impaired, but not abolished in response to aeroallergen. Moreover, the characteristic airways damage and hyperreactivity normally resulting from allergen inhalation were not attenuated. Induction of these structural and functional changes to the airways occurred in the absence of ovalbumin-specific IgE and IgG1, but IgG2a and IgG3 were detected in the sera of IL-4-deficient mice. CD4+ T cells isolated from both wild-type and IL-4-deficient mice given ovalbumin produced significant levels of IL-5 after in vitro stimulation. Treatment of IL-4-deficient mice with anti-IL-5 mAb before aeroallergen challenge abolished blood and airways eosinophilia, lung damage, and airways hyperreactivity. These results indicate that IL-4 is not essential for the development of IL-5-producing CD4+ T cells or for the induction of eosinophilic inflammation and airways damage and hyperreactivity. In response to sensitization and aerosol challenge, CD40-deficient mice did not produce ovalbumin-specific IgE, IgG isotypes, or IgA, and airways inflammation and hyperreactivity were not attenuated. Our results suggest that allergic airways disease can occur via pathways which operate independently of IL-4 and allergen-specific immunoglobulins. Activation of these pathways is intimately associated with IL-5 and eosinophilic inflammation. Such pathways may play a substantive role in the etiology of asthma.

Interleukin-4 (IL-4) promotes the growth of Th2-type cells while down regulating the development of Th1-type cells. It has been suggested that the actions of this factor inhibit Th1-type effector activity in vivo and may underlie the development of diseases normally controlled by cell-mediated immune responses. Here, we show that clearance of recombinant vaccinia viruses (VV) engineered to express the gene for murine IL-4 is markedly delayed in mice compared with control recombinant VV. While antiviral antibody levels and NK activity in mice given control virus or IL-4-expressing virus were similar, antiviral cytotoxic T-lymphocyte responses were profoundly suppressed throughout the course of infection with the latter. Limiting dilution analysis of IL-4-virus-infected spleens revealed a marked reduction in numbers of cytotoxic T-lymphocyte precursors. Furthermore, reverse transcriptase PCR analysis of splenic mRNA prepared from mice infected with the IL-4-expressing VV showed a marked down regulation of IL-12, gamma interferon, and IL-2 gene expression compared with that from mice given control virus. IL-4 also inhibited the production of nitric oxide (NO), a potent mediator of antimicrobial activity. Together, these data show that IL-4 markedly suppresses the development of antiviral cell-mediated immune responses in vivo with deleterious effects on virus clearance.