Anti-Interferon gamma Antikörper [MT126L] (A269859)

An effective AIDS vaccine should elicit strong humoral and cellular immune responses while maintaining low levels of CD4⁺ T-cell activation to avoid the generation of target cells for viral infection. The present study investigated two prime-boost regimens, both starting vaccination with single-cycle immunodeficiency virus, followed by two mucosal boosts with either recombinant adenovirus (rAd) or fowlpox virus (rFWPV) expressing SIVmac239 or SIVmac251 gag/pol and env genes, respectively. Finally, vectors were switched and systemically administered to the reciprocal group of animals. Only mucosal rFWPV immunizations followed by systemic rAd boost significantly protected animals against a repeated low-dose intrarectal challenge with pathogenic SIVmac251, resulting in a vaccine efficacy (i.e., risk reduction per exposure) of 68%. Delayed viral acquisition was associated with higher levels of activated CD8⁺ T cells and Gag-specific gamma interferon (IFN-?)-secreting CD8⁺ cells, low virus-specific CD4⁺ T-cell responses, and low Env antibody titers. In contrast, the systemic rFWPV boost induced strong virus-specific CD4⁺ T-cell activity. rAd and rFWPV also induced differential patterns of the innate immune responses, thereby possibly shaping the specific immunity. Plasma CXCL10 levels after final immunization correlated directly with virus-specific CD4⁺ T-cell responses and inversely with the number of exposures to infection. Also, the percentage of activated CD69⁺ CD8⁺ T cells correlated with the number of exposures to infection. Differential stimulation of the immune response likely provided the basis for the diverging levels of protection afforded by the vaccine regimen.IMPORTANCE A failed phase II AIDS vaccine trial led to the hypothesis that CD4⁺ T-cell activation can abrogate any potentially protective effects delivered by vaccination or promote acquisition of the virus because CD4⁺ T helper cells, required for an effective immune response, also represent the target cells for viral infection. We compared two vaccination protocols that elicited similar levels of Gag-specific immune responses in rhesus macaques. Only the animal group that had a low level of virus-specific CD4⁺ T cells in combination with high levels of activated CD8⁺ T cells was significantly protected from infection. Notably, protection was achieved despite the lack of appreciable Env antibody titers. Moreover, we show that both the vector and the route of immunization affected the level of CD4⁺ T-cell responses. Thus, mucosal immunization with FWPV-based vaccines should be considered a potent prime in prime-boost vaccination protocols.

Nonhuman primates can be used to study host immune responses to Mycobacterium tuberculosis Mauritian cynomolgus macaques (MCMs) are a unique group of animals that have limited major histocompatibility complex (MHC) genetic diversity, such that MHC-identical animals can be infected with M. tuberculosis Two MCMs homozygous for the relatively common M1 MHC haplotype were bronchoscopically infected with 41 CFU of the M. tuberculosis Erdman strain. Four other MCMs, which had at least one copy of the M1 MHC haplotype, were infected with a lower dose of 3 CFU M. tuberculosis All animals mounted similar T-cell responses to CFP-10 and ESAT-6. Two epitopes in CFP-10 were characterized, and the MHC class II alleles restricting them were determined. A third epitope in CFP-10 was identified but exhibited promiscuous restriction. The CFP-10 and ESAT-6 antigenic regions targeted by T cells in MCMs were comparable to those seen in cases of human M. tuberculosis infection. Our data lay the foundation for generating tetrameric molecules to study epitope-specific CD4 T cells in M. tuberculosis-infected MCMs, which may guide future testing of tuberculosis vaccines in nonhuman primates.

Background: The neotropical, non-human primates (NHP) of the genus Saimiri and Aotus are recommended by the World Health Organization as experimental models for the study of human malaria because these animals can be infected with the same Plasmodium that cause malaria in humans. However, one limitation is the lack of immunological tools to assess the immune response in these models. The present study focuses on the development and comparative use of molecular and immunological methods to evaluate the cellular immune response in Saimiri sciureus.

Methods: Blood samples were obtained from nineteen uninfected Saimiri. Peripheral blood mononuclear cells (PBMC) from these animals and splenocytes from one splenectomized animal were cultured for 6, 12, 18, 24, 48, 72 and 96 hrs in the presence of phorbol-12-myristate-13-acetate and ionomycin. The cytokine levels in the supernatant were detected using human and NHP cytometric bead array Th1/Th2 cytokine kits, the Bio-Plex Pro Human Cytokine Th1/Th2 Assay, enzyme-linked immunosorbent assay, enzyme-linked immunospot assays and intracellular cytokine secretion assays. Cytokine gene expression was examined through TaqMan® Gene Expression Real-Time PCR using predesigned human gene-specific primers and probes or primers and probes designed based on published S. sciureus cytokine sequences.

Results: The use of five assays based on monoclonal antibodies specific for human cytokines facilitated the detection of IL-2, IL-4 and/or IFN-?. TaqMan array plates facilitated the detection of 12 of the 28 cytokines assayed. However, only seven cytokines (IL-1A, IL-2, IL-10, IL-12B, IL-17, IFN-ß, and TNF) presented relative expression levels of at least 70% of the gene expression observed in human PBMC. The use of primers and probes specific for S. sciureus cytokines facilitated the detection of transcripts that showed relative expression below the threshold of 70%. The most efficient evaluation of cytokine gene expression, in PBMC and splenocytes, was observed after 6-12 hrs of culture, except for LTA in PBMC, whose expression was best analysed after 24 hrs of culture.

Conclusions: Real-time PCR facilitates the analysis of a large number of cytokines altered during malaria infection, and this technique is considered the best tool for the evaluation of the cellular immune response in S. sciureus.

Non-human primates (NHP) provide important animal models for studies on immune responses to infections and vaccines. When assessing cellular immunity in NHP, cytokines are almost exclusively analyzed utilizing cross-reactive anti-human antibodies. The functionality of antibodies has to be empirically established for each assay/application as well as NHP species. A rational approach was employed to identify monoclonal antibodies (mAb) cross-reactive with many NHP species. Panels of new and established mAbs against human Interferon (IFN)-? and Interleukin (IL)-2 were assessed for reactivity with eukaryotically expressed recombinant IFN-? and IL-2, respectively, from Old (rhesus, cynomolgus and pigtail macaques, African green monkey, sooty mangabey and baboon) and New World NHP (Ma's night monkey, squirrel monkey and common marmoset). Pan-reactive mAbs, recognizing cytokines from all NHP species, were further analyzed in capture assays and flow cytometry with NHP peripheral blood mononuclear cells (PBMC). Pan-reactive mAb pairs for IFN-? well as IL-2 were identified and used in ELISA to measure IFN-? and IL-2, respectively, in Old and New World NHP PBMC supernatants. The same mAb pairs displayed high functionality in ELISpot and FluoroSpot for the measurement of antigen-specific IFN-? and IL-2 responses using cynomolgus PBMC. Functionality of pan-reactive mAbs in flow cytometry was also verified with cynomolgus PBMC. The development of well-defined immunoassays functional with a panel of NHP species facilitates improved analyses of cellular immunity and enables inclusion in multiplex cytokine assays intended for a variety of NHP.

Introduction: Vascular endothelial growth factor (VEGF) is pivotal in tumor angiogenesis and therapies targeting the VEGF axis are widely used in the clinic for the treatment of cancer. We have developed a therapeutic vaccine targeting human (h)VEGF₁₆₅. hVEGF_26-104/RFASE is based on the truncated protein hVEGF_26-104 as antigen formulated in an oil-in-water emulsion containing the sulpholipopolysaccharide RFASE as adjuvant. Here we describe the toxicity and immunogenicity of this therapeutic vaccine in cynomolgus monkeys.

Methods: In total 54 cynomolgus monkeys were used and divided in 7 groups. Groups 1-3 were control groups, either receiving PBS alone (group 1), RFASE alone (group 2) or hVEGF_26-104 alone (group 3). Animals allocated to groups 4-7 received hVEGF_26-104 together with RFASE, but with varying doses of the antigen or the adjuvant. All animals were immunized four times with 2-week intervals and safety and immunogenicity were monitored until 3 days after the final immunization.

Results: Immunization induced an RFASE adjuvant dependent acute phase response. High titers of antibodies against hVEGF_26-104 and cross-reactive with hVEGF₁₆₅, were found in monkey sera, 28 days after primer immunization. These antibodies were able to inhibit the binding of the monoclonal antibody bevacizumab with hVEGF₁₆₅ in a competition ELISA. Moreover, the biological activity of hVEGF₁₆₅ could be inhibited by the addition of immunized monkey serum in a VEGF specific bioassay. Importantly, no adverse events commonly observed with VEGF neutralization were observed throughout the study.

Conclusion: These data show that hVEGF_26-104/RFASE can be safely administered in cynomolgus monkeys, induces the desired immune response and therefore support the clinical development of this vaccine.