Dengue virus neutralization by human immune sera: Role of envelope protein domain III-reactive antibody

Dengue viruses (DENV) are the etiological agents of dengue fever (DF) and dengue hemorrhagic fever (DHF). The DENV complex consists of four closely related viruses designated DENV serotypes 1 through-4. Although infection with one serotype induces cross reactive antibody to all 4 serotypes, the long term protective antibody response is restricted to the serotype responsible for infection. Cross-reactive antibodies appear to enhance infection during a second infection with a different serotype. The goal of the present study was to characterize the binding specificity and functional properties of human DENV immune sera. The study focused on domain III of the viral envelope protein (EDIII), as this region has a well characterized epitope that is recognized by strongly neutralizing serotype-specific mouse monoclonal antibodies (Mabs). Our results demonstrate that EDIII-reactive antibodies are present in primary and secondary DENV immune human sera. Human antibodies bound to a serotype specific epitope on EDIII after primary infection and a serotype cross reactive epitope on EDIII after secondary infection. However, EDIII-binding antibodies constituted only a small fraction of the total antibody in immune sera binding to DENV. Studies with complete and EDIII antibody depleted human immune sera demonstrated that EDIII binding antibodies play a minor role in DENV neutralization. We propose that human antibodies directed to other epitopes on the virus are primarily responsible for DENV neutralization. Our results have implications for understanding protective immunity following natural DENV infection and for evaluating DENV vaccines

Cutting Edge: NLRC5-Dependent Activation of the Inflammasome

The nucleotide-binding domain (NBD) leucine rich repeat (LRR) containing proteins, NLRs, are intracellular sensors of PAMPs and DAMPs. A subgroup of NLRs can form inflammasome complexes, which facilitate the maturation of pro-caspase-1 to caspase-1, leading to IL-1β and IL-18 cleavage and secretion. NLRC5 is predominantly expressed in hematopoetic cells and has not been studied for inflammasome function. RNAi-mediated knockdown of NLRC5 nearly eliminated caspase-1, IL-1β and IL-18 processing in response to bacterial infection, PAMPs and DAMPs. This was confirmed in primary human monocytic cells. NLRC5 together with procaspase-1, pro-IL-1β and the inflammasome adaptor, ASC, reconstituted inflammasome activity which showed cooperativity with NLPR3. The range of pathogens that activate NLRC5 inflammasome overlaps with those that activate NLRP3. Furthermore, NLRC5 biochemically associates with NLRP3 in an NBD-dependent but LRR-inhibitory fashion. These results invoke a model where NLRC5 interacts with NLRP3 to cooperatively activate the inflammasome

Natural Strain Variation and Antibody Neutralization of Dengue Serotype 3 Viruses

Dengue viruses (DENVs) are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs) have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII). It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge “type specific” epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII