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Polysaccharides (PS) have historically been viewed as T cell-independent antigens. However, in this paper I propose a new concept, based on substantial data from my laboratory over the previous 10 years, that during infections with PS-encapsulated extracellular bacteria, PS-specific IgG responses are largely CD4+ T cell dependent. Thus, capsular PS is typically encountered by the immune system covalently attached to the underlying subcapsular bacterial domain. I speculate that noncovalent association of PS with immunogenic proteins within the bacterial particle leads to recruitment of protein-specific CD4+ T cell help for the anti-PS response. However, differences in the composition and/or structure of the subcapsular domain of different extracellular bacteria may result in distinct anti-PS responses as well as differential effects on the immune response to coimmunizing soluble antigens. CD4+ T cell help for IgG anti-PS responses during infections with extracellular bacteria is likely to promote opsonic clearance of these rapidly growing pathogens. However, the expression of immunosuppressive components by certain bacteria may also serve to dampen such responses. [ABSTRACT FROM AUTHOR]

Chemotherapy or chemoradiotherapy conditioning regimens required for bone marrow transplantation (BMT) cause significant morbidity and mortality as a result of insufficient immune surveillance mechanisms leading to increased risks of infection and tumor recurrence. Such conditioning causes host stromal cell injury, impairing restoration of the central (thymus) and peripheral (spleen and lymph node) T cell compartments and slow immune reconstitution. The chemokine, CCL21, produced by host stromal cells, recruits T- and B-cells that provide lymphotoxin mediated instructive signals to stromal cells for lymphoid organogenesis. Moreover, T- and B-cell recruitment into these sites is required for optimal adaptive immune responses to pathogens and tumor antigens. Previously, we reported that CCL21 was markedly reduced in secondary lymphoid organs of transplanted animals. Here, we utilized adenoviral CCL21 gene transduced dendritic cells (DC/CCL21) given by footpad injections as a novel approach to restore CCL21 expression in secondary lymphoid organs post-transplant. CCL21 expression in secondary lymphoid organs reached levels of naïve controls and resulted in increased T cell trafficking to draining lymph nodes (LNs). An increase in both lymphoid tissue inducer cells and the B cell chemokine CXCL13 known to be important in LN formation was observed. Strikingly, only mice vaccinated with DC/CCL21 loaded with bacterial, viral or tumor antigens and not recipients of DC/control adenovirus loaded cells or no DCs had a marked increase in the systemic clearance of pathogens (bacteria; virus) and leukemia cells. Because DC/CCL21 vaccines have been tested in clinical trials for patients with lung cancer and melanoma, our studies provide the foundation for future trials of DC/CCL21 vaccination in patients receiving pre-transplant conditioning regimens. [ABSTRACT FROM AUTHOR]

In an effort to make affordable vaccines suitable for the regions most affected by HIV-1, we have constructed stable vaccines that express an HIV-1 subtype C mosaic Gag immunogen (BCG-GagM, MVA-GagM and DNA-GagM). Mosaic immunogens have been designed to address the tremendous diversity of this virus. Here we have shown that GagM buds from cells infected and transfected with MVA-GagM and DNA-GagM respectively and forms virus-like particles. Previously we showed that a BCG-GagM prime MVA-GagM boost generated strong cellular immune responses in mice. In this study immune responses to the DNA-GagM and MVA-GagM vaccines were evaluated in homologous and heterologous prime-boost vaccinations. The DNA homologous prime boost vaccination elicited predominantly CD8+ T cells while the homologous MVA vaccination induced predominantly CD4+ T cells. A heterologous DNA-GagM prime MVA-GagM boost induced strong, more balanced Gag CD8+ and CD4+ T cell responses and that were predominantly of an effector memory phenotype. The immunogenicity of the mosaic Gag (GagM) was compared to a naturally occurring subtype C Gag (GagN) using a DNA homologous vaccination regimen. DNA-GagN expresses a natural Gag with a sequence that was closest to the consensus sequence of subtype C viruses sampled in South Africa. DNA-GagM homologous vaccination induced cumulative HIV-1 Gag-specific IFN-γ ELISPOT responses that were 6.5-fold higher than those induced by the DNA-GagN vaccination. Similarly, DNA-GagM vaccination generated 7-fold higher levels of cytokine-positive CD8+ T cells than DNA-GagN, indicating that this subtype C mosaic Gag elicits far more potent immune responses than a consensus-type Gag. Cells transfected and infected with DNA-GagM and MVA-GagM respectively, expressed high levels of GagM and produced budding virus-like particles. Our data indicates that a heterologous prime boost regimen using DNA and MVA vaccines expressing HIV-1 subtype C mosaic Gag is highly immunogenic in mice and warrants further investigation in non-human primates. [ABSTRACT FROM AUTHOR]

The Bacille Calmette Guérin (BCG) vaccine is the only routine vaccination at birth that effectively induces neonatal T-helper 1 (Th1)-polarized immune responses. The primary cytokine that drives CD4+ T-cell Th1 differentiation is interleukin (IL)-12 p70, a heterodimeric cytokine composed of the IL-12 p35 and IL-12 p40 subunits. We therefore examined the mechanisms involved in BCG vaccine stimulation of IL-12 p35 and p40 production from human umbilical cord (neonatal) cells. We found that BCG bacilli did not upregulate IL-12 p35 mRNA production, but upregulated IL-12 p40 mRNA production in a Toll-like receptor (TLR)2-dependent manner, in human neonatal monocyte-derived dendritic cells (mdDCs). The combination of TLR2 signaling, Type I interferon (IFN), and Type II IFN induced maximal levels of IL-12 p35 and p40 mRNA production in human neonatal mdDCs. The cell-free supernatants of reconstituted BCG vaccine vials contained extracellular mycobacterial (BCG) DNA which could induce IFN-α (Type I IFN) production in human neonatal plasmacytoid dendritic cells (pDCs). BCG bacilli also stimulated human neonatal CD16lo natural killer (NK) cells to produce IFN-γ (Type II IFN) in a TLR2-dependent manner. We have therefore proposed a model where BCG vaccine could stimulate the combination of neonatal conventional DCs (cDCs), pDCs, and CD16lo NK cells to produce optimal neonatal IL-12 p35 and p40 (IL-12 p70) production and subsequent CD4+ T-cell Th1 polarization. An adjuvant that emulates the mechanism by which the BCG vaccine stimulates neonatal IL-12 p35 and p40 production could improve vaccine strategies at birth for protection against intracellular pathogens and toxins. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field. [ABSTRACT FROM AUTHOR]

Adenoviral vectored vaccines have shown considerable promise but could be improved by molecular adjuvants. Ligands in the TNF superfamily (TNFSF) are potential adjuvants for adenoviral vector (Ad5) vaccines based on their central role in adaptive immunity. Many TNFSF ligands require aggregation beyond the trimeric state (multi-trimerization) for optimal biological function. Here we describe Ad5 vaccines for HIV-1 Gag antigen (Ad5-Gag) adjuvanted with the TNFSF ligands 4-1BBL, BAFF, GITRL and CD27L constructed as soluble multi-trimeric proteins via fusion to Surfactant Protein D (SP-D) as a multimerization scaffold. Mice were vaccinated with Ad5-Gag combined with Ad5 expressing one of the SP-D-TNFSF constructs or single-chain IL-12p70 as adjuvant. To evaluate vaccine-induced protection, mice were challenged with vaccinia virus expressing Gag (vaccinia-Gag) which is known to target the female genital tract, a major route of sexually acquired HIV-1 infection. In this system, SP-D-4-1BBL or SP-D-BAFF led to significantly reduced vaccinia-Gag replication when compared to Ad5-Gag alone. In contrast, IL-12p70, SP-D-CD27L and SP-D-GITRL were not protective. Histological examination following vaccinia-Gag challenge showed a dramatic lymphocytic infiltration into the uterus and ovaries of SP-D-4-1BBL and SP-D-BAFF-treated animals. By day 5 post challenge, proinflammatory cytokines in the tissue were reduced, consistent with the enhanced control over viral replication. Splenocytes had no specific immune markers that correlated with protection induced by SP-D-4-1BBL and SP-D-BAFF versus other groups. IL-12p70, despite lack of anti-viral efficacy, increased the total numbers of splenic dextramer positive CD8+ T cells, effector memory T cells, and effector Gag-specific CD8+ T cells, suggesting that these markers are poor predictors of anti-viral immunity in this model. In conclusion, soluble multi-trimeric 4-1BBL and BAFF adjuvants led to strong protection from vaccinia-Gag challenge, but the protection was independent of standard immune markers. Soluble multi-trimeric SP-D-4-1BBL and SP-D-BAFF provide a novel technology to enhance adenoviral vector vaccines against HIV-1. [ABSTRACT FROM AUTHOR]

Our objective is to create gene immunogens targeted against drug-resistant HIV-1, focusing on HIV-1 enzymes as critical components in viral replication and drug resistance. Consensus-based gene vaccines are specifically fit for variable pathogens such as HIV-1 and have many advantages over viral genes and their expression-optimized variants. With this in mind, we designed the consensus integrase (IN) of the HIV-1 clade A strain predominant in the territory of the former Soviet Union and its inactivated derivative with and without mutations conferring resistance to elvitegravir. Humanized IN gene was synthesized; and inactivated derivatives (with 64D in the active site mutated to V) with and without elvitegravir-resistance mutations were generated by site-mutagenesis. Activity tests of IN variants expressed in E coli showed the consensus IN to be active, while both D64V-variants were devoid of specific activities. IN genes cloned in the DNA-immunization vector pVax1 (pVaxIN plasmids) were highly expressed in human and murine cell lines (>0.7 ng/cell). Injection of BALB/c mice with pVaxIN plasmids followed by electroporation generated potent IFN-γ and IL-2 responses registered in PBMC by day 15 and in splenocytes by day 23 after immunization. Multiparametric FACS demonstrated that CD8+ and CD4+ T cells of gene-immunized mice stimulated with IN-derived peptides secreted IFN-γ, IL-2, and TNF-α. The multi-cytokine responses of CD8+ and CD4+ T-cells correlated with the loss of in vivo activity of the luciferase reporter gene co-delivered with pVaxIN plasmids. This indicated the capacity of IN-specific CD4+ and CD8+ T-cells to clear IN/reporter co-expressing cells from the injection sites. Thus, the synthetic HIV-1 clade A integrase genes acted as potent immunogens generating polyfunctional Th1-type CD4+ and CD8+ T cells. Generation of such response is highly desirable for an effective HIV-1 vaccine as it offers a possibility to attack virus-infected cells via both MHC class I and II pathways. [ABSTRACT FROM AUTHOR]