Saravana Kanagavelu

Soluble multi-trimeric TNF superfamily ligand adjuvants enhance immune responses to a HIV-1 Gag DNA vaccine

BACKGROUND DNA vaccines remain an important component of HIV vaccination strategies, typically as part of a prime/boost vaccination strategy with viral vector or protein boost. A number of DNA prime/viral vector boost vaccines are currently being evaluated for both preclinical studies and in Phase I and Phase II clinical trials. These vaccines would benefit from molecular adjuvants that increase correlates of immunity during the DNA prime. While HIV vaccine immune correlates are still not well defined, there are a number of immune assays that have been shown to correlate with protection from viral challenge including CD8+ T cell avidity, antigen-specific proliferation, and polyfunctional cytokine secretion. METHODOLOGY AND PRINCIPAL FINDINGS Recombinant DNA vaccine adjuvants composed of a fusion between Surfactant Protein D (SP-D) and either CD40 Ligand (CD40L) or GITR Ligand (GITRL) were previously shown to enhance HIV-1 Gag DNA vaccines. Here we show that similar fusion constructs composed of the TNF superfamily ligands (TNFSFL) 4-1BBL, OX40L, RANKL, LIGHT, CD70, and BAFF can also enhanced immune responses to a HIV-1 Gag DNA vaccine. BALB/c mice were vaccinated intramuscularly with plasmids expressing secreted Gag and SP-D-TNFSFL fusions. Initially, mice were analyzed 2 weeks or 7 weeks following vaccination to evaluate the relative efficacy of each SP-D-TNFSFL construct. All SP-D-TNFSFL constructs enhanced at least one Gag-specific immune response compared to the parent vaccine. Importantly, the constructs SP-D-4-1BBL, SP-D-OX40L, and SP-D-LIGHT enhanced CD8+ T cell avidity and CD8+/CD4+ T cell proliferation 7 weeks post vaccination. These avidity and proliferation data suggest that 4-1BBL, OX40L, and LIGHT fusion constructs may be particularly effective as vaccine adjuvants. Constructs SP-D-OX40L, SP-D-LIGHT, and SP-D-BAFF enhanced Gag-specific IL-2 secretion in memory T cells, suggesting these adjuvants can increase the number of self-renewing Gag-specific CD8+ and/or CD4+ T cells. Finally adjuvants SP-D-OX40L and SP-D-CD70 increased T(H)1 (IgG2a) but not T(H)2 (IgG1) antibody responses in the vaccinated animals. Surprisingly, the B cell-activating protein BAFF did not enhance anti-Gag antibody responses when given as an SP-D fusion adjuvant, but nonetheless enhanced CD4+ and CD8+ T cell responses. CONCLUSIONS We present evidence that various SP-D-TNFSFL fusion constructs can enhance immune responses following DNA vaccination with HIV-1 Gag expression plasmid. These data support the continued evaluation of SP-D-TNFSFL fusion proteins as molecular adjuvants for DNA and/or viral vector vaccines. Constructs of particular interest included SP-D-OX40L, SP-D-4-1BBL, SP-D-LIGHT, and SP-D-CD70. SP-D-BAFF was surprisingly effective at enhancing T cell responses, despite its inability to enhance anti-Gag antibody secretion.

Preclinical evaluation of HIV-1 therapeutic ex vivo dendritic cell vaccines expressing consensus Gag antigens and conserved Gag epitopes

BACKGROUND Dendritic cell (DC) therapy is a promising technology for the treatment of HIV infected individuals. HIV-1 Gag- and Nef RNA-loaded DC have previously been shown to induce immune responses ex vivo following coculture with autologous lymphocytes. However, polyfunctionality and memory responses following coculture have not been evaluated. In addition, little is known regarding whether specific HIV-1 proteome components, such as highly conserved regions of the HIV-1, could enhance clinical responses following DC therapy. METHODOLOGY AND PRINCIPAL FINDINGS To determine the breadth of the immune responses to antigen loaded DC, we analyzed polyfunctional T cell response ex vivo to Gag RNA loaded DC. Blood samples were used to generate monocyte derived DC, which were then matured and cocultured with autologous lymphocytes. We found that cytokine-matured DC loaded with Gag RNA was able to induce Gag-specific IFN-γ and IL-2 responses after a 12-day coculture. We characterized these responses by polyfunctional intracellular cytokine staining and evaluation of T cell memory phenotypes. Central memory CD8+ T cells were induced ex vivo after DC coculture from each of 3 patients, and the effector memory pool was increased by DC coculture from 2 patients. We also observed a decrease in the terminal effector and intermediate CD8+ T cell pool and an increase in the naïve/other population. There was a reduction in terminal effector and intermediate CD4+ T cells, and a corresponding increase in naïve/other CD4+ T cells. Finally, we evaluated conserved regions of Gag as a novel DC therapy immunogen and found that a conserved element (CE) p24 Gag antigen elicited IFN-γ and IL-2 responses comparable to those induced by a full-length Gag antigen. CONCLUSIONS We showed that RNA-loaded DC therapy induced a polyfunctional T cell response ex vivo, supporting the use of such DC-therapy for HIV infection. However, the central and effector memory phenotypes of T cells did not appear to be enhanced during coculture with Gag RNA-loaded DC. Furthermore, comparable antigen-specific responses were induced in HIV infected individuals using full-length Gag or only conserved elements of the Gag p24 protein. This indicates that immune responses can be focused onto the conserved elements of Gag in the absence of other Gag components.

Human intestinal epithelial cells express interleukin-10 through Toll-like receptor 4-mediated epithelial-macrophage crosstalk

In the intestine, interaction between epithelial cells and macrophages (MΦs) create a unique immunoregulatory microenvironment necessary to maintain local immune and tissue homeostasis. Human intestinal epithelial cells (IECs) have been shown to express interleukin (IL)-10, which keeps epithelial integrity. We have demonstrated that bacterial signaling through Toll-like receptor (TLR) 4 induces 15-deoxy-Δ-12,14-prostaglandin J2 (15d-PGJ2) synthesis in intestinal MΦs by cyclooxygenase (Cox)-2 expression. Here, we show that TLR4 signaling generates crosstalk between IECs and MΦs that enhances IL-10 expression in IECs. Direct stimulation of TLR4 leads to the expression of IL-10 in IECs, while the presence of MΦs in a Transwell system induces another peak in IL-10 expression in IECs at a later time point. The second peak of the IL-10 expression is two times greater than the first peak. This late induction of IL-10 depends on the nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ that is accumulated in IECs by TLR4-mediated inhibition of the ubiquitin-proteasomal pathway. TLR4 signaling in MΦs in turn synthesizes 15d-PGJ2 through p38 and ERK activation and Cox-2 induction, which activates PPARγ in IECs. These results suggest that TLR4 signaling maintains IL-10 production in IECs by generating epithelial-MΦs crosstalk, which is an important mechanism in the maintenance of intestinal homeostasis mediated through host-bacterial interactions.

A unique host defense pathway: TRIF mediates both antiviral and antibacterial immune responses

Both anti-viral and anti-bacterial host defense mechanisms involve TRIF signaling. TRIF provides early clearance of pathogens and coordination of a local inflammatory ensemble through an interferon cascade, while it may trigger organ damage. The multipotentiality of TRIF-mediated immune machinery may direct the fate of our continuous battle with microbes.

DNA vaccine molecular adjuvants SP-D-BAFF and SP-D-APRIL enhance anti-gp120 immune response and increase HIV-1 neutralizing antibody titers

ABSTRACT Broadly neutralizing antibodies (bNAbs) specific for conserved epitopes on the HIV-1 envelope (Env) are believed to be essential for protection against multiple HIV-1 clades. However, vaccines capable of stimulating the production of bNAbs remain a major challenge. Given that polyreactivity and autoreactivity are considered important characteristics of anti-HIV bNAbs, we designed an HIV vaccine incorporating the molecular adjuvants BAFF (B cell activating factor) and APRIL (a proliferation-inducing ligand) with the potential to facilitate the maturation of polyreactive and autoreactive B cells as well as to enhance the affinity and/or avidity of Env-specific antibodies. We designed recombinant DNA plasmids encoding soluble multitrimers of BAFF and APRIL using surfactant protein D as a scaffold, and we vaccinated mice with these molecular adjuvants using DNA and DNA-protein vaccination strategies. We found that immunization of mice with a DNA vaccine encoding BAFF or APRIL multitrimers, together with interleukin 12 (IL-12) and membrane-bound HIV-1 Env gp140, induced neutralizing antibodies against tier 1 and tier 2 (vaccine strain) viruses. The APRIL-containing vaccine was particularly effective at generating tier 2 neutralizing antibodies following a protein boost. These BAFF and APRIL effects coincided with an enhanced germinal center (GC) reaction, increased anti-gp120 antibody-secreting cells, and increased anti-gp120 functional avidity. Notably, BAFF and APRIL did not cause indiscriminate B cell expansion or an increase in total IgG. We propose that BAFF and APRIL multitrimers are promising molecular adjuvants for vaccines designed to induce bNAbs against HIV-1. IMPORTANCE Recent identification of antibodies that neutralize most HIV-1 strains has revived hopes and efforts to create novel vaccines that can effectively stimulate HIV-1 neutralizing antibodies. However, the multiple immune evasion properties of HIV have hampered these efforts. These include the instability of the gp120 trimer, the inaccessibility of the conserved sequences, highly variable protein sequences, and the loss of HIV-1-specific antibody-producing cells during development. We have shown previously that tumor necrosis factor (TNF) superfamily ligands, including BAFF and APRIL, can be multitrimerized using the lung protein SP-D (surfactant protein D), enhancing immune responses. Here we show that DNA or DNA-protein vaccines encoding BAFF or APRIL multitrimers, IL-12p70, and membrane-bound HIV-1 Env gp140 induced tier 1 and tier 2 neutralizing antibodies in a mouse model. BAFF and APRIL enhanced the immune reaction, improved antibody binding, and increased the numbers of anti-HIV-1 antibody-secreting cells. Adaptation of this vaccine design may prove useful in designing preventive HIV-1 vaccines for humans.

HIV-1 Adenoviral Vector Vaccines Expressing Multi-Trimeric BAFF and 4-1BBL Enhance T Cell Mediated Anti-Viral Immunity

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.

EBV LMP1, a viral mimic of CD40, activates dendritic cells and functions as a molecular adjuvant when incorporated into an HIV vaccine.

HIV‐1 does not significantly activate cellular immunity, which has made it difficult to use attenuated forms of HIV‐1 as a vaccine. In contrast, EBV induces robust T cell responses in most infected individuals, perhaps as this virus contains LMP1, a viral mimic of CD40, which is a key activating molecule for DCs and macrophages. Consequently, studies were conducted using LMP1 and LMP1‐CD40, a related construct formed by replacing the intracellular signaling domain of LMP1 with that of CD40. Upon electroporation into DCs, LMP1 and LMP1‐CD40 mRNAs were sufficient to up‐regulate costimulatory molecules and proinflammatory cytokines, indicating that these molecules can function in isolation as adjuvant‐like molecules. As a first step toward an improved HIV vaccine, LMP1 and LMP1‐CD40 were introduced into a HIV‐1 construct to produce virions encoding these proteins. Transduction of DCs and macrophages with these viruses induced morphological changes and up‐regulated costimulatory molecules and cytokine production by these cells. HIV‐LMP1 enhanced the antigen‐presenting function of DCs, as measured in an in vitro immunization assay. Taken together, these data show that LMP1 and LMP1‐CD40 are portable gene cassettes with strong adjuvant properties that can be introduced into viruses such as HIV, which by themselves, are insufficient to induce protective cellular immunity.

In Vivo Effects of Lattice Radiation Therapy on Local and Distant Lung Cancer: Potential Role of Immunomodulation

Radiation is a potent immune-modulator that elicits cell death upon tumor, stromal and angiogenic compartments of tumor microenvironment. Here, we test a novel approach of high-dose radiation delivery using three dimensional volume based lattice radiation therapy (LRT) to understand the impact of different volume irradiation in eliciting both local and metastatic/distant tumor control through modulation of tumor immune micro-environment. To study such effects of LRT, tumors were implanted in both hind legs of C57BL/6 mice using Lewis lung carcinoma 1 (LLC1) cells. Mice were divided into five groups: untreated; partial tumor volume groups included two 10% vertices, one 20% vertex and one 50% vertex of the total tumor volume; and 100% open-field irradiation. Tumors implanted in the left flank were irradiated with a single dose of 20 Gy while the tumors in the right flank were unirradiated. Tumor growth and regression as well as immune responses (such as Th1 and Th2; T-cell infiltration) were determined after radiation treatment. Results demonstrated that both 100% open-field irradiation and 20% volume irradiation (in two 10% volumes) resulted in significant growth delay in the irradiated tumor. Further, all types of radiation exposures, partial or 100% volume, demonstrated distal effectiveness, however, 20% volume irradiation (in two 10% volumes) and 50% tumor volume irradiation led to maximum growth delay. Mice treated with partial tumor volume radiation induced a robust IFN-γ and Th1 response when compared to whole-tumor irradiation and down-modulated Th2 functions. The presence of increased CD3+ cells and TRAIL in partially irradiated tumor volumes correlated well with tumor growth delay. Further, serum obtained from any of the LRT treated mice caused growth inhibition of endothelial cells when compared to serum obtained from either untreated or open-field irradiated groups. These results indicate that high-dose partial volume irradiation can cause an improved distant effect than the total tumor volume irradiation through activating the host immune system.

TIR-domain-containing adapter-inducing interferon-β (TRIF) regulates Th17-mediated intestinal immunopathology in colitis

Gastrointestinal mucosa reserves abundant Th17 cells where host response to commensal bacteria maintains Th17-cell generation. Although functional heterogeneity and dynamic plasticity of Th17 cells appear to be involved in chronic inflammatory disorders, how their plasticity is regulated in intestinal mucosa is unknown. Here we show that innate TRIF signaling regulates intestinal Th17-cell generation and plasticity during colitis. Absence of TRIF in mice resulted in increased severity of experimental colitis, which was associated with aberrant generation of Th17 cells especially of interferon (IFN)-γ-expressing Th17 cells in the lamina propria. The abnormal generation and plasticity of Th17 cells involved impaired expression of interleukin (IL)-27p28 by lamina propria macrophages but not dendritic cells. Treatment of TRIF-deficient mice with IL-27p28 during colitis reduced the number and IFN-γ expression of Th17 cells in the intestine. In vitro, TRIF-deficient macrophages induced more Th17 cells than wild-type (WT) macrophages during co-culture with WT naive T cells in response to cecal bacterial antigen. Many of Th17 cells induced by TRIF-deficient macrophages expressed IFN-γ due to impaired expression of IL-27p28 by macrophages and defective activation of STAT1 in T cells. These results outline TRIF-dependent regulatory mechanism by which host response to intestinal bacteria maintains Th17-cell-mediated pathology during colitis.

Systemic Activation of TLR3-Dependent TRIF Signaling Confers Host Defense against Gram-Negative Bacteria in the Intestine.

Recognition of Gram-negative bacteria by toll-like receptor (TLR)4 induces MyD88 and TRIF mediated responses. We have shown that TRIF-dependent responses play an important role in intestinal defense against Gram-negative enteropathogens. In the current study, we examined underlying mechanisms of how systemic TRIF activation enhances intestinal immune defense against Gram-negative bacteria. First we confirmed that the protective effect of poly I:C against enteric infection of mice with Yersinia enterocolitica was dependent on TLR3-mediated TRIF signaling by using TLR3-deficient mice. This protection was unique in TRIF-dependent TLR signaling because systemic stimulation of mice with agonists for TLR2 (Pam3CSK4) or TLR5 (flagellin) did not reduce mortality on Y. enterocolitica infection. Systemic administration of poly I:C mobilized CD11c+, F4/80+, and Gr−1hi cells from lamina propria and activated NK cells in the mesenteric lymph nodes (MLN) within 24 h. This innate immune cell rearrangement was type I IFN dependent and mediated through upregulation of TLR4 followed by CCR7 expression in these innate immune cells found in the intestinal mucosa. Poly I:C induced IFN-γ expression by NK cells in the MLN, which was mediated through type I IFNs and IL-12p40 from antigen presenting cells and consequent activation of STAT1 and STAT4 in NK cells. This formation of innate immunity significantly contributed to the elimination of bacteria in the MLN. Our results demonstrated an innate immune network in the intestine that can be established by systemic stimulation of TRIF, which provides a strong host defense against Gram-negative pathogens. The mechanism underlying TRIF-mediated protective immunity may be useful to develop novel therapies for enteric bacterial infection.