Induction of mucosal immune responses against a heterologous antigen fused to filamentous hemagglutinin after intranasal immunization with recombinant Bordetella pertussis.

Live vaccine vectors are usually very effective and generally elicit immune responses of higher magnitude and longer duration than nonliving vectors. Consequently, much attention has been turned to the engineering of oral pathogens for the delivery of foreign antigens to the gut-associated lymphoid tissues. However, no bacterial vector has yet been designed to specifically take advantage of the nasal route of mucosal vaccination. Herein we describe a genetic system for the expression of heterologous antigens fused to the filamentous hemagglutinin (FHA) in Bordetella pertussis. The Schistosoma mansoni glutathione S-transferase (Sm28GST) fused to FHA was detected at the cell surface and in the culture supernatants of recombinant B. pertussis. The mouse colonization capacity and autoagglutination of the recombinant microorganism were indistinguishable from those of the wild-type strain. In addition, and in contrast to the wild-type strain, a single intranasal administration of the recombinant strain induced both IgA and IgG antibodies against Sm28GST and against FHA in the bronchoalveolar lavage fluids. No anti-Sm28GST antibodies were detected in the serum, strongly suggesting that the observed immune response was of mucosal origin. This demonstrates, to our knowledge, for the first time that recombinant respiratory pathogens can induce mucosal immune responses against heterologous antigens, and this may constitute a first step toward the development of combined live vaccines administrable via the respiratory route.

Human neoplasms elicit multiple specific immune responses in the autologous host.

Expression of cDNA libraries from human melanoma, renal cancer, astrocytoma, and Hodgkin disease in Escherichia coli and screening for clones reactive with high-titer IgG antibodies in autologous patient serum lead to the discovery of at least four antigens with a restricted expression pattern in each tumor. Besides antigens known to elicit T-cell responses, such as MAGE-1 and tyrosinase, numerous additional antigens that were overexpressed or specifically expressed in tumors of the same type were identified. Sequence analyses suggest that many of these molecules, besides being the target of a specific immune response, might be of relevance for tumor growth. Antibodies to a given antigen were usually confined to patients with the same tumor type. The unexpected frequency of human tumor antigens, which can be readily defined at the molecular level by the serological analysis of autologous tumor cDNA expression cloning, indicates that human neoplasms elicit multiple specific immune responses in the autologous host and provides diagnostic and therapeutic approaches to human cancer.

Protective immune responses induced by secretion of a chimeric soluble protein from a recombinant Mycobacterium bovis bacillus Calmette-Guérin vector candidate vaccine for human immunodeficiency virus type 1 in small animals.

A recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) vector-based vaccine that secretes the V3 principal neutralizing epitope of human immunodeficiency virus (HIV) could induce immune response to the epitope and prevent the viral infection. By using the Japanese consensus sequence of HIV-1, we successfully constructed chimeric protein secretion vectors by selecting an appropriate insertion site of a carrier protein and established the principal neutralizing determinant (PND)-peptide secretion system in BCG. The recombinant BCG (rBCG)-inoculated guinea pigs were initially screened by delayed-type hypersensitivity (DTH) skin reactions to the PND peptide, followed by passive transfer of the DTH by the systemic route. Further, immunization of mice with the rBCG resulted in induction of cytotoxic T lymphocytes. The guinea pig immune antisera showed elevated titers to the PND peptide and neutralized HIVMN, and administration of serum IgG from the vaccinated guinea pigs was effective in completely blocking the HIV infection in thymus/liver transplanted severe combined immunodeficiency (SCID)/hu or SCID/PBL mice. In addition, the immune serum IgG was shown to neutralize primary field isolates of HIV that match the neutralizing sequence motif by a peripheral blood mononuclear cell-based virus neutralization assay. The data support the idea that the antigen-secreting rBCG system can be used as a tool for development of HIV vaccines.

Oxidative/reductive conjugation of mannan to antigen selects for T1 or T2 immune responses.

The induction of CD8+ cytotoxic T lymphocytes (CTLs) is desirable for immunization against many diseases, and recombinant-synthetic peptide antigens are now favored agents to use. However, a major problem is how to induce CTLs, which requires a T1-type response to such synthetic antigens. We report that T1-type (generating high CTL, low antibody) or T2-type (the reciprocal) responses can be induced by conjugation of the antigen to the carbohydrate polymer mannan: T1 responses are selected by using oxidizing conditions; T2 responses are selected by using reducing conditions for the conjugation. Using human MUC1 as a model antigen in mice, immunization with oxidized mannan-MUC1 fusion protein (ox-M-FP) led to complete tumor protection (challenge up to 5 x 10(7) MUC1+ tumor cells), CTLs, and a high CTL precursor (CTLp) frequency (1/6900), whereas immunization with reduced mannan-MUC1 FP (red-M-FP) led to poor protection after challenge with only 10(6) MUC1+ tumor cells, no CTLs, and a low CTLp frequency (1/87,800). Ox-M-FP selects for a T1 response (mediated here by CD8+ cells) with high interferon gamma (IFN-gamma) secretion, no interleukin 4 (IL-4), and a predominant IgG2a antibody response; red-M-FP selects for a T2-type response with IL-4 production and a high predominant IgG1 antibody response but no IFN-gamma.

Characterization of a Leishmania tropica antigen that detects immune responses in Desert Storm viscerotropic leishmaniasis patients.

A chronic debilitating parasitic infection, viscerotropic leishmaniasis (VTL), has been described in Operation Desert Storm veterans. Diagnosis of this disease, caused by Leishmania tropica, has been difficult due to low or absent specific immune responses in traditional assays. We report the cloning and characterization of two genomic fragments encoding portions of a single 210-kDa L. tropica protein useful for the diagnosis of VTL in U.S. military personnel. The recombinant proteins encoded by these fragments, recombinant (r) Lt-1 and rLt-2, contain a 33-amino acid repeat that reacts with sera from Desert Storm VTL patients and with sera from L. tropica-infected patients with cutaneous leishmaniasis. Antibody reactivities to rLt-1 indicated a bias toward IgG2 in VTL patient sera. Peripheral blood mononuclear cells from VTL patients produced interferon gamma, but not interleukin 4 or 10, in response to rLt-1. No cytokine production was observed in response to parasite lysate. The results indicate that specific leishmanial antigens may be used to detect immune responses in VTL patients with chronic infections.

Mucosal and systemic immune responses to a recombinant protein expressed on the surface of the oral commensal bacterium Streptococcus gordonii after oral colonization.

To circumvent the need to engineer pathogenic microorganisms as live vaccine-delivery vehicles, a system was developed which allowed for the stable expression of a wide range of protein antigens on the surface of Gram-positive commensal bacteria. The human oral commensal Streptococcus gordonii was engineered to surface express a 204-amino acid allergen from hornet venom (Ag5.2) as a fusion with the anchor region of the M6 protein of Streptococcus pyogenes. The immunogenicity of the M6-Ag5.2 fusion protein was assessed in mice inoculated orally and intranasally with a single dose of recombinant bacteria, resulting in the colonization of the oral/pharyngeal mucosa for 10-11 weeks. A significant increase of Ag5.2-specific IgA with relation to the total IgA was detected in saliva and lung lavages when compared with mice colonized with wild-type S. gordonii. A systemic IgG response to Ag5.2 was also induced after oral colonization. Thus, recombinant Gram-positive commensal bacteria may be a safe and effective way of inducing a local and systemic immune response.

Human anti-idiotypic antibodies induced a humoral and cellular immune response against a colorectal carcinoma-associated antigen in patients.

Induction of immunity against antigens expressed on tumor cells might prevent or delay recurrence of the disease. Six patients operated on for colorectal carcinoma were immunized with human monoclonal anti-idiotypic antibodies (h-Ab2) against the mouse 17-1A anti-colon carcinoma antibody, mimicking a nominal antigen (GA733-2). All patients developed a long-lasting T-cell immunity against the extracellular domain of GA733-2 (GA733-2E) (produced in a baculovirus system) and h-Ab2. This was shown in vitro by specific cell proliferation (DNA-synthesis) assay as well as by interleukin 2 and interferon gamma production and in vivo by the delayed-type hypersensitivity reaction. Five patients mounted a specific humoral response (IgG) against the tumor antigen GA733-2E (ELISA) and tumor cells expressing GA733-2. Epitope mapping using 23 overlapping peptides of GA733-2E revealed that the B-cell epitope was localized close to the N terminus of GA733-2. Binding of the antibodies to the tumor antigen and to one 18-aa peptide was inhibited by h-Ab2, indicating that the antibodies were able to bind to the antigen as well as to h-Ab2. The results suggest that our h-Ab2 might be able to induce an anti-tumor immunity which may control the growth of tumor cells in vivo.

Role of IFN-gamma in immune responses to Candida albicans infections

Candida albicans is the most frequent etiologic agent that causes opportunistic fungal infections called candidiasis, a disease whose systemic manifestation could prove fatal and whose incidence is increasing as a result of an expanding immunocompromised population. Here we review the role of interferon-gamma (IFN-γ) in host protection against invasive candidiasis. This cytokine plays an essential role in both the innate and adaptive arms of the immune response to candidiasis. We focus on recent progress on host-pathogen interactions leading to the production of IFN-γ by host cells. IFN-γ is produced by CD4 Th1, CD8, γδ T, and natural killer (NK) cells, essentially in response to both IL-12 and/or IL-18; more recently, a subset of C. albicans-specific Th17 cells have been described to produce both IL-17 and IFN-γ. IFN-γ plays an important role in the regulation of the immune system as well as in the control of the infectious process, as it is required for optimal activation of phagocytes, collaborates in the generation of protective antibody response, and favors the development of a Th1 protective response.

Peroxisomes are platforms for cytomegalovirus’ evasion from the cellular immune response

The human cytomegalovirus developed distinct evasion mechanisms from the cellular antiviral response involving vMIA, a virally-encoded protein that is not only able to prevent cellular apoptosis but also to inhibit signalling downstream from mitochondrial MAVS. vMIA has been shown to localize at mitochondria and to trigger their fragmentation, a phenomenon proven to be essential for the signalling inhibition. Here, we demonstrate that vMIA is also localized at peroxisomes, induces their fragmentation and inhibits the peroxisomal-dependent antiviral signalling pathway. Importantly, we demonstrate that peroxisomal fragmentation is not essential for vMIA to specifically inhibit signalling downstream the peroxisomal MAVS. We also show that vMIA interacts with the cytoplasmic chaperone Pex19, suggesting that the virus has developed a strategy to highjack the peroxisomal membrane proteins' transport machinery. Furthermore, we show that vMIA is able to specifically interact with the peroxisomal MAVS. Our results demonstrate that peroxisomes constitute a platform for evasion of the cellular antiviral response and that the human cytomegalovirus has developed a mechanism by which it is able to specifically evade the peroxisomal MAVS-dependent antiviral signalling.

Systemic and local immune responses in sheep after Neospora caninum experimental infection at early, mid and late gestation.

Besides its importance in cattle, Neospora caninum may also pose a high risk as abortifacient for small ruminants. We have recently demonstrated that the outcome of experimental infection of pregnant sheep with 10(6) Nc-Spain7 tachyzoites is strongly dependent on the time of gestation. In the current study, we assessed peripheral and local immune response in those animals. Serological analysis revealed earlier and higher IFN-γ and IgG responses in ewes infected at early (G1) and mid (G2) gestation, when abortion occurred. IL-4 was not detected in sera from any sheep. Inflammatory infiltrates in the placenta mainly consisted of CD8+ and, to a lesser extent, CD4+ T cells and macrophages (CD163+). The infiltrate was more intense in sheep infected at mid-gestation. In the foetal mesenchyme, mostly free tachyzoites were found in animals infected at G1, while those infected in G2 displayed predominantly particulate antigen, and parasitophorous vacuoles were detected in sheep infected at G3. A similar pattern of placental cytokine mRNA expression was found in all groups, displaying a strengthened upregulation of IFN-γ and IL-4 and milder increases of TNF-α and IL-10, reminiscent of a mixed Th1 and Th2 response. IL-12 and IL-6 were only slightly upregulated in G2, and TGF-β was downregulated in G1 and G2, suggestive of limited T regulatory (Treg) cell activity. No significant expression of TLR2 or TLR4 could be detected. In summary, this study confirms the pivotal role of systemic and local immune responses at different times of gestation during N. caninum infection in sheep.

Quantitative Association Tests of Immune Responses to Antigens of Mycobacterium Tuberculosis: A Study of Twins in West Africa

There is now considerable evidence that host genetic factors are important in determining the outcome of infection with Mycobacterium tuberculosis (MTB). The aim of this study was to assess the role of several candidate genes in the variation observed in the immune responses to MTB antigens. In-vitro assays of T-cell proliferation, an in-vivo intradermal delayed hypersensitivity response; cytokine and antibody secretions to several mycobacterial peptide antigens were assessed in healthy, but exposed, West African twins. Candidate gene polymorphisms were typed in the NRAMP1, Vitamin D receptor, IL10, IL4, IL4 receptor and CTLA-4 genes. Variants of the loci IL10 (-1082 G/A), CTLA-4 (49 A/G) and the IL4 receptor (128 A/G) showed significant associations with immune responses to several antigens. T-cell proliferative responses and antibody responses were reduced, TNF-alpha responses were increased for subjects with the CTLA-4 G allele. The T-cell proliferative responses of subjects with IL10 GA and GG genotypes differed significantly. IL4 receptor AG and GG genotypes also showed significant differences in their T-cell proliferative responses to MTB antigens. These results yield a greater understanding of the genetic mechanisms that underlie the immune responses in tuberculosis and have implications for the design of therapeutic interventions.

HPV-16 L1 VLP vaccine elicits a broad-spectrum of cytokine responses in whole blood

Here, we evaluated innate and adaptive immune system cytokine responses induced by HPV-16 L1 VLP in whole blood (WB) cultures from individuals receiving the vaccine (n = 20) or placebo (n = 4) before and after vaccination. 11 cytokines were measured: IL- 1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, 1L- 10, IL- 12, IFN-gamma, TNF-alpha, and GM-CSF using multiplex bead arrays. Cytokine profiles from WB samples clearly discriminated between vaccine and placebo recipients and between pre and post-vaccination responses. Significant increases in Th1, Th2 and inflammatory cytokines were observed in WB assays following vaccination. Results from WB assays were compared against parallel PBMC-based assays in a subset of patients. Differences between whole blood assay and PBMC were observed, with the highest levels of induction found for WB for several cytokines. Our results indicate that multiplex assays for cytokine profiling in WB are an efficient toot for assessing broad spectrum, innate and adaptive immune responses to vaccines and identifying immunologic correlates of protection in efficacy studies. (c) 2005 Elsevier Ltd. All rights reserved.