A 12-Month-Interval Dosing Study in Adults Indicates That a Single Dose of the National Institute of Allergy and Infectious Diseases Tetravalent Dengue Vaccine Induces a Robust Neutralizing Antibody Response.

UNLABELLED: The ideal dengue vaccine will provide protection against all serotypes of dengue virus and will be economical and uncomplicated in its administration. To determine the ability of a single dose of the live attenuated tetravalent dengue vaccine TV003 to induce a suitable neutralizing antibody response, a placebo-controlled clinical trial was performed in 48 healthy adults who received 2 doses of vaccine or placebo administered 12 months apart. Evaluation of safety, vaccine viremia, and neutralizing antibody response after each dose indicated that the first dose of vaccine was capable of preventing infection with the second dose, thus indicating that multiple doses are unnecessary. CLINICAL TRIALS REGISTRATION: NCT01782300.

Robust and Balanced Immune Responses to All 4 Dengue Virus Serotypes Following Administration of a Single Dose of a Live Attenuated Tetravalent Dengue Vaccine to Healthy, Flavivirus-Naive Adults.

BACKGROUND: The 4 serotypes of dengue virus, DENV-1-4, are the leading cause of arboviral disease globally. The ideal dengue vaccine would provide protection against all serotypes after a single dose. METHODS: Two randomized, placebo-controlled trials were performed with 168 flavivirus-naive adults to demonstrate the safety and immunogenicity of a live attenuated tetravalent dengue vaccine (TV003), compared with those of a second tetravalent vaccine with an enhanced DENV-2 component (TV005), and to evaluate the benefit of a booster dose at 6 months. Safety data, viremia, and neutralizing antibody titers were evaluated. RESULTS: A single dose of TV005 elicited a tetravalent response in 90% of vaccinees by 3 months after vaccination and a trivalent response in 98%. Compared with TV003, the higher-dose DENV-2 component increased the observed frequency of immunogenicity to DENV-2 in the TV005 trial. Both the first and second doses were well tolerated. Neither vaccine viremia, rash, nor a significant antibody boost were observed following a second dose. CONCLUSIONS: A single subcutaneous dose of TV005 dengue vaccine is safe and induces a tetravalent antibody response at an unprecedented frequency among vaccinees. A second dose has limited benefit and appears to be unnecessary. Studies to confirm these findings and assess vaccine efficacy will now move to populations in regions where DENV transmission is endemic. CLINICAL TRIALS REGISTRATION: NCT01072786 and NCT01436422.

Fluorochrome-linked immunoassay for functional analysis of the mannose binding lectin complement pathway to the level of C3 cleavage.

The humoral response to invading pathogens is mediated by a repertoire of innate immune molecules and receptors able to recognize pathogen-associated molecular patterns. Mannose binding lectin (MBL) and ficolins are initiation molecules of the lectin complement pathway (LCP) that bridge innate and adaptive immunity. Activation of the MBL-dependent lectin pathway, to the level of C3 cleavage, requires functional MASP-2, C2, C4 and C3, all of which have been identified with genetic polymorphisms that can affect protein concentration and function. Current assays for MBL and MASP-2 lack the ability to assess activation of all components to the level of C3 cleavage in a single assay platform. We developed a novel, low volume, fluorochrome linked immunoassay (FLISA) that quantitatively assesses the functional status of MBL, MASP-2 and C3 convertase in a single well. The assay can be used with plasma or serum. Multiple freeze/thaw cycles of serum do not significantly alter the assay, making it ideal for high throughput of large sample databases with minimal volume use. The FLISA can be used potentially to identify specific human disease correlations between these components and clinical outcomes in already established databases.

Role of the alternative pathway in the early complement activation following major trauma.

Complement activation has been reported after major trauma. However, little is known about the clinical relevance and the mechanisms of complement activation early after trauma. Therefore, the aim of this study was to measure complement activation, to identify the roles of injury severity and hypoperfusion, to determine the predominant activated pathway, and to identify the clinical significance of early complement activation in trauma patients. A total of 208 adult trauma patients were enrolled in this prospective single-center cohort study of major trauma patients. Blood samples were obtained within 30 min after injury before any significant fluid resuscitation. Complement (C5b-9) was activated early after trauma, correlated with injury severity and tissue hypoperfusion, and was associated with increased mortality rate and with the development of organ failure such as acute lung injury and acute renal failure. The alternative pathway seems to be the predominant activated complement pathway early after trauma. However, the classical and/or the lectin pathway initiated complement activation because of the correlation between plasma levels of C4d and C3a/C5b-9. Finally, in patients with low C3a levels, C5b-9 levels correlated with plasma levels of prothrombin fragments 1 + 2, a marker of thrombin generation, suggesting additional C3-independent complement activation by thrombin after severe trauma. In summary, complement activation via its amplification by the alternative pathway is observed early after trauma and correlates with injury severity, tissue hypoperfusion, and worse clinical outcomes. Besides complement activation by the classical and/or lectin pathways, there is an independent association between thrombin generation and complement activation.

Mannose-binding lectin binds IgM to activate the lectin complement pathway in vitro and in vivo.

Recent evidence has implicated a role for the MBL-dependent lectin pathway in gastrointestinal and myocardial ischemia/reperfusion (I/R)-induced injury. However, previous studies have implicated IgM and the classical pathway as initiators of complement activation following I/R. Thus, we investigated the potential interaction between MBL and IgM leading to complement activation. Using surface plasmon resonance, we demonstrate that MBL does bind human IgM. Subsequently, functional complement activation was demonstrated in vitro following sensitization of human RBCs with mouse anti-human CD59 IgM and more lysis was observed with MBL sufficient sera compared to MBL deficient (KO) sera. Similarly, treatment of human endothelial cells with mouse anti-human CD59 IgM, MBL and MASP-2 activated and deposited C4. These data suggest that the presence of both IgM and MBL can activate the lectin pathway in vitro. Serum ALT levels increased significantly in sIgM/MBL-A/C KO mice reconstituted with WT plasma compared to sIgM/MBL-A/C KO mice reconstituted with MBL-A/C KO plasma following gastrointestinal (G) I/R. Similarly, intestinal C3 deposition was greater in sIgM/MBL-A/C KO mice reconstituted with WT plasma compared to sIgM/MBL-A/C KO mice treated with MBL-A/C KO plasma. These data indicate for the first time that both IgM and MBL-A/C are required for GI/R-induced complement activation and subsequent injury.

Enhanced antigen-specific antibody and cytokine responses when targeting antigen to human FcGAMMA receptor type I using an anti-human FcGAMMA receptor type I-streptavidin fusion protein in an adjuvant-free system.

There is a continuing need for alternatives to current human adjuvants. Recombinant protein vaccines, which target antigen to human Fc gamma receptor type I (hFcgammaRI) on hFcgammaRI-expressing antigen presenting cells, provide one potential alternative. Using a recombinant anti-hFcgammaRI-antigen fusion protein and adjuvant independent mouse model, we demonstrate enhanced antigen-specific antibody responses to low doses of antigen, when targeting antigen to hFcgammaRI in vivo. Enhanced antibody production to hFcyRI-targeted antigen is evident in both primary and secondary immune responses, as compared to that of non-targeted antigen. Furthermore, antibody isotype and cytokine responses following immunization with hFcgammaRI-targeted antigen, suggest enhancement of both Th1 and Th2 responses.

Mannose-binding lectin is a regulator of inflammation that accompanies myocardial ischemia and reperfusion injury.

The mannose-binding lectin (MBL), a circulating pattern recognition molecule, recognizes a wide range of infectious agents with resultant initiation of the complement cascade in an Ab-independent manner. MBL recognizes infectious non-self and altered self in the guise of apoptotic and necrotic cells. In this study, we demonstrate that mice lacking MBL, and hence are devoid of MBL-dependent lectin pathway activation but have fully active alternative and classical complement pathways, are protected from cardiac reperfusion injury with resultant preservation of cardiac function. Significantly, mice that lack a major component of the classical complement pathway initiation complex (C1q) but have an intact MBL complement pathway, are not protected from injury. These results suggest that the MBL-dependent pathway of complement activation is a key regulator of myocardial reperfusion ischemic injury. MBL is an example of a pattern recognition molecule that plays a dual role in modifying inflammatory responses to sterile and infectious injury.

Initiation of complement activation following oxidative stress. In vitro and in vivo observations.

Ischemia and reperfusion of organs/tissues induce a state of inflammation that can lead to tissue injury. Focus on development of effective therapeutics based on sound pre-clinical work and the role of leukocytes in models of human disease has not lead to a successful clinical trial for anti-leukocyte technologies. For the past >30 years, it has been known that complement activation plays a role in the inflammation and tissue injury associated with ischemia/reperfusion (I/R) injury. In the last 10 years, several complement inhibitors have made their way from the bench to bedside. Will a complement inhibitor eventually be approved for clinical treatment of I/R type diseases? What pathway(s) are involved in I/R injury, and what role do they play? What specific complement components are needed for resolution of inflammation and what components need to be inhibited to decrease tissue injury? This short review will focus on the current state of the art knowledge about complement, complement pathways, complement components and several promising clinical biologics that inhibit complement activation. This review is not a complete review of complement in ischemia/reperfusion injury, but it raises important questions about the role of complement, its pathways and the current knowledge in the area of ischemia/reperfusion injury.

Generation of a human IgG3-streptavidin fusion protein. Implications for the inhibition and elimination of auto-reactive B cells.

Antibodies against self-molecules play a significant role in the development and progression of Systemic Lupus Erythematosus, as well as a number of other autoimmune disorders. Immunosuppressive drugs have been used to control this process. However, they are normally not specific to the offending cell, and can actually suppress beneficial immune responses to pathogens. In this paper a genetically engineered targeting molecule is described, which has the capacity to target antigen-specific B cells for inhibition or elimination. The targeting molecule is a fusion of streptavidin subunit to the constant region of human IgG3 (IgG3-Av). It is demonstrated by ELISA and flow cytometry that IgG3-Av binds biotinylated antigen as well as human Fc gamma receptors present on myeloid cells. It is also shown by confocal microscopy and flow cytometry, that IgG3-Av can mediate Fc receptor-dependent phagocytosis of latex microspheres adsorbed with biotinylated antigen. Furthermore, the IgG3-Av construct can modulate Ca++ flux, characteristic of B cell inhibition as well as ADCC of B cells in an antigen-specific manner. In summary, these studies describe an approach, which has the potential to be used as a treatment to inhibit or remove antigen-specific (auto-reactive) B cells.

A two-component modular approach for enhancing T-cell activation utilizing a unique anti-FcgammaRI-streptavidin construct and microspheres coated with biotinylated-antigen.

The professional antigen presenting cell (APC) plays an essential role in the initiation and propagation of the acquired immune response. Thus, much work has been done in designing strategies that target vaccine antigen (Ag) to APC. Utilizing recombinant DNA technology, we have created a unique two-component system that delivers biotinylated Ag to the Fc gamma receptor type I (FcgammaRI) on APC. Our studies demonstrate that we can successfully engineer FcgammaRI-specific targeting element proteins that simultaneously bind both biotin and recognize FcgammaRI. Additionally, we are able to engineer biotinylated Ag, which form functional elements when adsorbed onto latex microspheres. Furthermore, the targeting and functional element components bind to each other and successfully form two-component immunogens. T-cell activation in response to targeted Ag-laden microspheres is 10- to 100-fold greater than the response to the non-targeted Ag-laden microspheres. This enhancement is 100- to 1000-fold greater than the responses generated to soluble Ag. Thus, our results suggest that specific targeting of Ag-laden microspheres to FcgammaRI may significantly enhance the adjuvant properties of microparticulate delivery systems. Further development of this system may help to elucidate the mechanisms involved in generating enhanced responses to APC-targeted vaccines and significantly advance vaccine technology.