Therapeutic efficacy of a potent anti-Venezuelan equine encephalitis virus antibody is contingent on Fc effector function.

The development of specific, safe, and potent monoclonal antibodies (Abs) has led to novel therapeutic options for infectious disease. In addition to preventing viral infection through neutralization, Abs can clear infected cells and induce immunomodulatory functions through engagement of their crystallizable fragment (Fc) with complement proteins and Fc receptors on immune cells. Little is known about the role of Fc effector functions of neutralizing Abs in the context of encephalitic alphavirus infection. To determine the role of Fc effector function in therapeutic efficacy against Venezuelan equine encephalitis virus (VEEV), we compared the potently neutralizing anti-VEEV human IgG F5 (hF5) Ab with intact Fc function (hF5-WT) or containing the loss of function Fc mutations L234A and L235A (hF5-LALA) in the context of VEEV infection. We observed significantly reduced binding to complement and Fc receptors, as well as differential in vitro kinetics of Fc-mediated cytotoxicity for hF5-LALA compared to hF5-WT. The in vivo efficacy of hF5-LALA was comparable to hF5-WT at -24 and + 24 h post infection, with both Abs providing high levels of protection. However, when hF5-WT and hF5-LALA were administered + 48 h post infection, there was a significant decrease in the therapeutic efficacy of hF5-LALA. Together these results demonstrate that optimal therapeutic Ab treatment of VEEV, and possibly other encephalitic alphaviruses, requires neutralization paired with engagement of immune effectors via the Fc region.

Relationship between HIV-1 Gag Multimerization and Membrane Binding.

HIV-1 viral particle assembly occurs specifically at the plasma membrane and is driven primarily by the viral polyprotein Gag. Selective association of Gag with the plasma membrane is a key step in the viral assembly pathway, which is traditionally attributed to the MA domain. MA regulates specific plasma membrane binding through two primary mechanisms including: (1) specific interaction of the MA highly basic region (HBR) with the plasma membrane phospholipid phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2], and (2) tRNA binding to the MA HBR, which prevents Gag association with non-PI(4,5)P2 containing membranes. Gag multimerization, driven by both CA-CA inter-protein interactions and NC-RNA binding, also plays an essential role in viral particle assembly, mediating the establishment and growth of the immature Gag lattice on the plasma membrane. In addition to these functions, the multimerization of HIV-1 Gag has also been demonstrated to enhance its membrane binding activity through the MA domain. This review provides an overview of the mechanisms regulating Gag membrane binding through the MA domain and multimerization through the CA and NC domains, and examines how these two functions are intertwined, allowing for multimerization mediated enhancement of Gag membrane binding.

Molecular Determinants in tRNA D-arm Required for Inhibition of HIV-1 Gag Membrane Binding.

Plasma-membrane-specific localization of Gag, an essential step in HIV-1 particle assembly, is regulated by the interaction of the Gag MA domain with PI(4,5)P2 and tRNA-mediated inhibition of non-specific or premature membrane binding. Different tRNAs inhibit PI(4,5)P2-independent membrane binding to varying degrees in vitro; however, the structural determinants for this difference remain unknown. Here we demonstrate that membrane binding of full-length Gag synthesized in vitro using reticulocyte lysates is inhibited when RNAs that contain the anticodon arm of tRNAPro, but not that of tRNALys3, are added exogenously. In contrast, in the context of a liposome binding assay in which the effects of tRNAs on purified MA were tested, full-length tRNALys3 showed greater inhibition of MA membrane binding than full-length tRNAPro. While transplantation of the D loop sequence of tRNALys3 into tRNAPro resulted in a modest increase in the inhibitory effect relative to WT tRNAPro, replacing the entire D arm sequence with that of tRNALys3 was necessary to confer the full inhibitory effects upon tRNAPro. Together, these results demonstrate that the D arm of tRNALys3 is a major determinant of strong inhibition of MA membrane binding and that this inhibitory effect requires not only the D loop, which was recently reported to contact the MA highly basic region, but the loop sequence in the context of the D arm structure.

The first case of severe acute hemolytic transfusion reaction caused by anti-Sc2.

BACKGROUND: Alloantibodies to the low-frequency antigen Scianna-2 (Sc2) have been implicated in cases of hemolytic disease of the fetus and newborn but never in hemolytic transfusion reactions (HTRs); thus, the clinical significance of anti-Sc2 has yet to be fully addressed. STUDY DESIGN AND METHODS: A 26-year-old woman with thalassemia presented rigors, fever, nausea, abdominal pain, and hemolytic biochemistry after exposure to 75 mL of plasma-reduced red blood cells (RBCs). The RBC unit was issued by electronic crossmatch but was 3+ incompatible on recrossmatch by gel indirect antiglobulin test (IAT). The patient had anti-Sc2 previously identified, but considered to be clinically insignificant. The transfusion history was reviewed and a monocyte monolayer assay (MMA) was performed. RESULTS: The patient was investigated for a RBC reaction 9 years prior, when she developed symptoms of HTR. The RBC unit was crossmatched by immediate spin due to consistent screen negativity. Full crossmatch found the RBC 1+ incompatible by gel IAT with both pre/post samples, while direct antiglobulin test was negative (pre) and 1+ immunoglobulin G positive (post). The antibody remained unidentified and she was committed to gel IAT crossmatch. Two-years later, the specificity to Sc2 was deduced when one RBC unit was found 3+ incompatible. Finally, the transfusion reaction reported herein occurred when she received by happenstance RBCs from the same donor who was associated with the remote reaction 9 years earlier. MMA yielded highly positive phagocytic indices only for Sc2+ RBCs, including the donor's RBCs that triggered the severe HTR. CONCLUSION: This is the first case of HTR caused by anti-Sc2 confirmed by clinical findings and MMA.

Siderophore vaccine conjugates protect against uropathogenic Escherichia coli urinary tract infection.

Uropathogenic Escherichia coli (UPEC) is the primary cause of uncomplicated urinary tract infections (UTIs). Whereas most infections are isolated cases, 1 in 40 women experience recurrent UTIs. The rise in antibiotic resistance has complicated the management of chronic UTIs and necessitates new preventative strategies. Currently, no UTI vaccines are approved for use in the United States, and the development of a highly effective vaccine remains elusive. Here, we have pursued a strategy for eliciting protective immunity by vaccinating with small molecules required for pathogenesis, rather than proteins or peptides. Small iron-chelating molecules called siderophores were selected as antigens to vaccinate against UTI for this vaccine strategy. These pathogen-associated stealth siderophores evade host immune defenses and enhance bacterial virulence. Previous animal studies revealed that vaccination with siderophore receptor proteins protects against UTI. The poor solubility of these integral outer-membrane proteins in aqueous solutions limits their practical utility. Because their cognate siderophores are water soluble, we hypothesized that these bacterial-derived small molecules are prime vaccine candidates. To test this hypothesis, we immunized mice with siderophores conjugated to an immunogenic carrier protein. The siderophore-protein conjugates elicited an adaptive immune response that targeted bacterial stealth siderophores and protected against UTI. Our study has identified additional antigens suitable for a multicomponent UTI vaccine and highlights the potential use of bacterial-derived small molecules as antigens in vaccine therapies.