Cytokine Profile of Children Hospitalized with Virologically-Confirmed Dengue during Two Phase III Vaccine Efficacy Trials.

BACKGROUND: Two large-scale efficacy studies with the recombinant yellow fever-17D-dengue virus, live-attenuated, tetravalent dengue vaccine (CYD-TDV) candidate undertaken in Asia (NCT01373281) and Latin America (NCT01374516) demonstrated significant protection against dengue disease during two years' active surveillance (active phase). Long-term follow up of participants for breakthrough disease leading to hospitalization is currently ongoing (hospital phase). METHODOLOGY/PRINCIPAL FINDINGS: We assessed the cytokine profile in acute sera from selected participants hospitalized (including during the active phase) up to the beginning of the second year of long-term follow up for both studies. The serum concentrations of 38 cytokines were measured in duplicate using the Milliplex Human Cytokine MAGNETIC BEAD Premixed 38 Plex commercial kit (Millipore, Billerica, MA, USA). Partial least squares discriminant analyses did not reveal any difference in the overall cytokine profile of CYD-TDV and placebo recipients hospitalized for breakthrough dengue regardless of stratification used. In addition, there was no difference in the cytokine profile for breakthrough dengue among those aged <9 years versus those aged ≥ 9 years. CONCLUSIONS/SIGNIFICANCE: These exploratory findings show that CYD-TDV does not induce a particular immune profile versus placebo, corroborating the clinical profile observed.

Assessment of bivalent and tetravalent dengue vaccine formulations in flavivirus-naïve adults in Mexico.

UNLABELLED: Several ChimeriVax-Dengue (CYD)-based vaccination strategies were investigated as potential alternatives to vaccination with tetravalent CYD vaccine (CYD-TDV) in this phase IIa trial conducted in 2008-9 in 150 healthy adults. Participants were randomized and vaccinated on D0 and D105 (± 15 days). One group received bivalent CYD vaccine against serotypes 1 and 3 (CYD-1;3) on day 0 and CYD-2;4 on day 105 (± 15 days). Two groups received an injection at each timepoint of a tetravalent blend of CYD-1;3;4 and a VERO cell derived, live attenuated vaccine against serotype 2 (VDV-2), or the reference CYD-TDV. A fourth group received Japanese encephalitis (JE) vaccine on days -14, -7 and 0, followed by CYD-TDV on day 105. Viraemia was infrequent in all groups. CYD-4 viraemia was most frequent after tetravalent vaccination, while CYD-3 viraemia was most frequent after the first bivalent vaccination. Immunogenicity as assessed by 50% plaque reduction neutralisation test on D28 was comparable after the first injection of either tetravalent vaccine, and increased after the second injection, particularly with the blended CYD-1;3;4/ VDV-2 vaccine. In the bivalent vaccine group, immune response against serotype 3 was highest and the second injection elicited a low immune response against CYD 2 and 4. Immune responses after the first injection of CYD-TDV in the JE-primed group were in general higher than after the first injection in the other groups. All tested regimens were well tolerated without marked differences between groups. Bivalent vaccination showed no advantage in terms of immunogenicity. CLINICAL TRIAL REGISTRATION NUMBER: NCT00740155.

Persistence of Th1/Tc1 responses one year after tetravalent dengue vaccination in adults and adolescents in Singapore.

To characterize the cell mediated immunity (CMI) induced by the investigational CYD tetravalent dengue vaccine (TDV), we developed a whole-blood, intracellular cytokine staining (ICS) assay and a multiplex assay, each requiring 3 mL of blood. We assessed CMI before and 28 d after a first and third injection of CYD-TDV and one year after the third injection in a subset of 80 adolescents and adults enrolled in a phase II trial in Singapore (ClinicalTrial.gov NCT NCT00880893). CD4/IFNγ/TNFα responses specific to dengue NS3 were detected before vaccination. Vaccination induced YF-17D-NS3-specific CD8/IFNγ responses, without significant TNFα, and a CYD-specific Th1/Tc1 cellular response in all participants, which was characterized by predominant IFNγ secretion compared with TNFα, associated with low level IL-13 secretion in multiplex analysis of peripheral blood mononuclear cells (PBMC) supernatants after restimulation with each the CYD vaccine viruses. Responses were directed mainly against CYD-4 after the first vaccination, and were more balanced against all four serotypes after the third vaccination. The same qualitative profile was observed one year after the third vaccination, with approximately 2-fold lower NS3-specific responses, and 3-fold lower serotype-specific cellular responses. These findings confirm previous observations regarding both the nature and specificity of cellular responses induced by CYD-TDV, and for the first time demonstrate the persistence of cellular responses after one year. We also established the feasibility of analyzing CMI with small blood samples, allowing such analysis to be considered for pediatric trials.

Gene profiling analysis of ALVAC infected human monocyte derived dendritic cells.

The recombinant canarypox virus ALVAC is being extensively studied as vaccine vector for the development of new vaccine strategies against chronic infectious diseases and cancer. However, the mechanisms by which ALVAC initiates the immune response have not been completely elucidated. In order to determine the type of innate immunity triggered by ALVAC, we characterized the gene expression profile of human monocyte derived dendritic cells (MDDCs) upon ALVAC infection. These cells are permissive to poxvirus infection and play a key role in the initiation of immune responses. The majority of the genes that were up-regulated by ALVAC belong to the type I interferon signaling pathway including IRF7, STAT1, RIG-1, and MDA-5. Genes involved in the NF-kappaB pathway were not up-regulated. The gene encoding for the chemokine CXCL10, a direct target of the transcription factor IRF3 was among those up-regulated and DC secretion of CXCL10 following exposure to ALVAC was confirmed by ELISA. Many downstream type I interferon activated genes with anti-viral activity (PKR, Mx, ISG15 and OAS among others) were also up-regulated in response to ALVAC. Among these, ISG15 expression in its unconjugated form by Western blot analysis was demonstrated. In view of these results we propose that ALVAC induces type I interferon anti-viral innate immunity via a cytosolic pattern-recognition-receptor (PRR) sensing double-stranded DNA, through activation of IRF3 and IRF7. These findings may aid in the design of more effective ALVAC-vectored vaccines.

AlphaPIX Rho GTPase guanine nucleotide exchange factor regulates lymphocyte functions and antigen receptor signaling.

AlphaPIX is a Rho GTPase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletal-membrane complexes. It has been shown that PIX proteins play roles in some immune cells, including neutrophils and T cells. In this study, we report the immune system phenotype of alphaPIX knockout mice. We extended alphaPIX expression experiments and found that whereas alphaPIX was specific to immune cells, its homolog betaPIX was expressed in a wider range of cells. Mice lacking alphaPIX had reduced numbers of mature lymphocytes and defective immune responses. Antigen receptor-directed proliferation of alphaPIX(-) T and B cells was also reduced, but basal migration was enhanced. Accompanying these defects, formation of T-cell-B-cell conjugates and recruitment of PAK and Lfa-1 integrin to the immune synapse were impaired in the absence of alphaPIX. Proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of PAK and expression of GIT2 in both T cells and B cells. These results reveal specific roles for alphaPIX in the immune system and suggest that redundancy with betaPIX precludes a more severe immune phenotype.

The Canarypox-virus vaccine vector ALVAC triggers the release of IFN-gamma by Natural Killer (NK) cells enhancing Th1 polarization.

We investigated the mechanism by which ALVAC activates innate immunity. Combining ALVAC with protein antigens significantly augmented antigen-specific IgG2a responses; this was dependent on the presence of bioactive interferon (IFN)-gamma. Immuno-depletion of NK cells prior to ALVAC immunisation abrogated IFN-gamma production indicating that they are the main cellular source of early IFN-gamma in vivo. Murine bone-marrow derived dendritic cells (BMDCs) cultured in the presence of ALVAC secreted high levels of the chemokines CXCL10 and CCL2 and up-regulated expression of the maturation markers CD40, CD80 and CD86. Therefore, we conclude that ALVAC acts as an adjuvant through a mechanism requiring NK cell derived IFN-gamma, DC activation and chemokine secretion.

The Lsc RhoGEF mediates signaling from thromboxane A2 to actin polymerization and apoptosis in thymocytes.

The Lsc RhoGEF (also known as p115-RhoGEF) is a GTP exchange factor (GEF), an activator of GTPases of the Rho family. Lsc has a RhoGEF domain specific for Rho GTPase and a regulator of G protein signaling (RGS) domain specific for Galpha(12/13) subunits. One G protein receptor that can couple to Galpha(12/13) subunits is the receptor for thromboxane A(2 )(TXA(2)), thromboxane-prostanoid (called TP), which is highly expressed in immature thymocytes. TXA(2) has been implicated in thymocyte apoptosis. We found that Lsc(-/-) mice on a BALB/c background show thymic hyperplasia due to increased numbers of thymocytes and that these numbers further increase with the age of the mice. To investigate a role for Lsc in TXA(2) signaling, we analyzed activation of primary thymocytes by TXA(2) in vitro. TXA(2)-induced apoptosis of double-positive thymocytes and Rho activation required Lsc, and TXA(2) stimulation of actin polymerization and cofilin phosphorylation required both Lsc and Rho kinase (ROCK). Additionally, in the absence of Lsc, phosphorylation of the survival kinase Akt in response to TXA(2) was greatly enhanced. Together, these data demonstrate that Lsc is essential for mediating TXA(2 )signaling involved in apoptosis and actin organization and suggest that TXA(2) regulates thymic cellularity via Lsc.