We have shown previously that the betagamma subunits of the heterotrimeric G proteins regulate the organization of the pericentriolarly localized Golgi stacks. In this report, evidence is presented that the downstream target of Gbetagamma is protein kinase D (PKD), an isoform of protein kinase C. PKD, unlike other members of this class of serine/threonine kinases, contains a pleckstrin homology (PH) domain. Our results demonstrate that Gbetagamma directly activates PKD by interacting with its PH domain. Inhibition of PKD activity through the use of pharmacological agents, synthetic peptide substrates, and, more specifically, the PH domain of PKD prevents Gbetagamma-mediated Golgi breakdown. Our findings suggest a possible mechanism by which the direct interaction of Gbetagamma with PKD regulates the dynamics of Golgi membranes and protein secretion.

Antibody neutralization is an important component of protective immunity against vaccinia virus (VACV).Two distinct virion forms, mature virion and enveloped virion (MV and EV, respectively), possess separate functions and nonoverlapping immunological properties. In this study we examined the mechanics of EV neutralization, focusing on EV protein B5 (also called B5R). We show that neutralization of EV is predominantly complement dependent. From a panel of high-affinity anti-B5 monoclonal antibodies (MAbs), the only potent neutralizer in vitro (90% at 535 ng/ml) was an immunoglobulin G2a (IgG2a), and neutralization was complement mediated. This MAb was the most protective in vivo against lethal intranasal VACV challenge. Further studies demonstrated that in vivo depletion of complement caused a >50% loss of anti-B5 IgG2a protection, directly establishing the importance of complement for protection against the EV form. However, the mechanism of protection is not sterilizing immunity via elimination of the inoculum as the viral inoculum consisted of a purified MV form. The prevention of illness in vivo indicated rapid control of infection. We further demonstrate that antibody-mediated killing of VACV-infected cells expressing surface B5 is a second protective mechanism provided by complement-fixing anti-B5 IgG. Cell killing was very efficient, and this effector function was highly isotype specific. These results indicate that anti-B5 antibody-directed cell lysis via complement is a powerful mechanism for clearance of infected cells, keeping poxvirus-infected cells from being invisible to humoral immune responses. These findings highlight the importance of multiple mechanisms of antibody-mediated protection against VACV and point to key immunobiological differences between MVs and EVs that impact the outcome of infection.Vaccines are one of the most cost-effective medical treatments in modern civilization (80). A smallpox vaccine was the first human vaccine, and the modern smallpox vaccine, live vaccinia virus (VACV), is the most successful human vaccine, bringing about the worldwide eradication of smallpox disease due to a heroic World Health Organization campaign in the 1960s and 1970s (27). Elucidating the immunobiology underlying the protection provided by the smallpox vaccine will continue to reveal vaccinology principles that can be applied to future vaccine development against other infectious scourges (95). Neutralizing antibodies are of primary importance in the protection from smallpox provided by the smallpox vaccine in animal models (6,25,33,64) and humans (4). Vaccinia immune globulin (VIG) has been shown to be an effective treatment against smallpox (45) as it was able to reduce the number of smallpox cases by ϳ80% among exposed individuals in four case-controlled studies (42,45,50,51,66).Neutralizing antibodies confer protection mainly through the recognition of structures on the surface of virus particles, and therefore antiviral antibodies directed against the surface of virions are of primary inter...

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Vaccinia virus possesses two immunologically distinct virion forms in vivo-mature virion (MV, IMV) and extracellular virion (EV, EEV). Here we show that combination therapy with two fully human mAbs against an immunodominant MV antigen, H3 (H3L), and an EV antigen, B5 (B5R), provides significantly better protection against vaccinia infection in a small animal model of progressive vaccinia (SCID mice infected with VACV NYCBOH vaccine strain) than a single human monoclonal or human vaccinia immune globulin (VIG), the currently licensed therapeutic for side effects of smallpox vaccination.

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A proof-of-concept study evaluating the potential of Streptococcus pneumoniae Pneumococcal Surface Protein A (PspA) as a passive immunization target was conducted. We describe the generation and isolation of several broadly reactive mouse anti-PspA monoclonal antibodies (mAbs). MAb 140H1 displayed (i) 98% strain coverage, (ii) activity in complement deposition and opsonophagocytic killing (OPK) assays, which are thought to predict the in vivo efficacy of anti-pneumococcal mAbs, (iii) efficacy in mouse sepsis models both alone and in combination with standard-of-care antibiotics, and (iv) therapeutic activity in a mouse pneumonia model. Moreover, we demonstrate that antibody engineering can significantly enhance anti-PspA mAb effector function. We believe that PspA has promising potential as a target for the therapy of invasive pneumococcal disease by mAbs, which could be used alone or in conjunction with standard-of-care antibiotics.

The contributions of this author are as follows: conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper. Dr. Cécile Martin should also be included in the Funding section. The correct Funding information is as follows: This work was supported by Kyowa Hakko Kirin Co., Ltd. Japan (http://www.kyowakirin.com/index.html). All named authors are or were employees of Kyowa Kirin Pharmaceutical Research, Inc. or Kyowa Hakko Kirin Co., Ltd., Japan at the time of their involvement in the reported study. Kyowa Hakko Kirin Co., Ltd., Japan provided funding for the research of the study and provided support in the form of salaries for authors SK, TO, SB, RC, BG, TK, GD, CM, JL, LK, TT, and YK, but no individuals employed by the funder, other than the named authors, had any additional role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section.