Glass Capillary Tube Feeding: A Method for Infecting Nymphal Ixodes scapularis (Acari: Ixodidae) with the Lyme Disease Spirochete Borrelia burgdorferi

We evaluated an artificial capillary feeding method to infect nymphal Ixodes scapularis (Say) ticks with Borrelia burgdorferi, the causative agent of Lyme disease. Thirty to 70% of the nymphs were infected after feeding for 2.5 h from glass capillary tubes filled with a solution of spirochetes. Capillary infection was stable and persisted in the nymphs for at least 10 d after feeding. Capillary feeding also maintained natural vector competence patterns because I. scapularis ticks acquired infection unlike Dermacentor variablis (Say), which did not become infected. Capillary infected I. scapularis nymphs were capable of transmitting the infection to naive mice although not as efficiently as naturally infected nymphs. The capillary infection method is convenient and is a better alternative to syringe inoculation as a means of infecting animals with B. burgdorferi

Rapid, directed transport of DC-SIGN clusters in the plasma membrane

C-type lectins, including dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN), are all-purpose pathogen receptors that exist in nanoclusters in plasma membranes of dendritic cells. A small fraction of these clusters, obvious from the videos, can undergo rapid, directed transport in the plane of the plasma membrane at average speeds of more than 1 μm/s in both dendritic cells and MX DC-SIGN murine fibroblasts ectopically expressing DC-SIGN. Surprisingly, instantaneous speeds can be considerably greater. In MX DC-SIGN cells, many cluster trajectories are colinear with microtubules that reside close to the ventral membrane, and the microtubule-depolymerizing drug, nocodazole, markedly reduced the areal density of directed movement trajectories, suggesting a microtubule motor–driven transport mechanism; by contrast, latrunculin A, which affects the actin network, did not depress this movement. Rapid, retrograde movement of DC-SIGN may be an efficient mechanism for bringing bound pathogen on the leading edge and projections of dendritic cells to the perinuclear region for internalization and processing. Dengue virus bound to DC-SIGN on dendritic projections was rapidly transported toward the cell center. The existence of this movement within the plasma membrane points to an unexpected lateral transport mechanism in mammalian cells and challenges our current concepts of cortex-membrane interactions

In Vitro Assembly and Stabilization of Dengue and Zika Virus Envelope Protein Homo-Dimers

Zika virus (ZIKV) and the 4 dengue virus (DENV) serotypes are mosquito-borne Flaviviruses that are associated with severe neuronal and hemorrhagic syndromes. The mature flavivirus infectious virion has 90 envelope (E) protein homo-dimers that pack tightly to form a smooth protein coat with icosahedral symmetry. Human antibodies that strongly neutralize ZIKV and DENVs recognize complex quaternary structure epitopes displayed on E-homo-dimers and higher order structures. The ZIKV and DENV E protein expressed as a soluble protein is mainly a monomer that does not display quaternary epitopes, which may explain the modest success with soluble recombinant E (sRecE) as a vaccine and diagnostic antigen. New strategies are needed to design recombinant immunogens that display these critical immune targets. Here we present two novel methods for building or stabilizing in vitro E-protein homo-dimers that display quaternary epitopes. In the first approach we immobilize sRecE to enable subsequent dimer generation. As an alternate method, we describe the use of human mAbs to stabilize homo-dimers in solution. The ability to produce recombinant E protein dimers displaying quaternary structure epitopes is an important advance with applications in flavivirus diagnostics and vaccine development

Preexisting Neutralizing Antibody Responses Distinguish Clinically Inapparent and Apparent Dengue Virus Infections in a Sri Lankan Pediatric Cohort

Dengue viruses (DENVs) are mosquito-borne flaviviruses that infect humans. The clinical presentation of DENV infection ranges from inapparent infection to dengue hemorrhagic fever and dengue shock syndrome. We analyzed samples from a pediatric dengue cohort study in Sri Lanka to explore whether antibody responses differentiated clinically apparent infections from clinically inapparent infections. In DENV-naive individuals exposed to primary DENV infections, we observed no difference in the quantity or quality of acquired antibodies between inapparent and apparent infections. Children who experienced primary infections had broad, serotype–cross-neutralizing antibody responses that narrowed in breadth to a single serotype over a 12-month period after infection. In DENV immune children who were experiencing a repeat infection, we observed a strong association between preexisting neutralizing antibodies and clinical outcome. Notably, children with preexisting monospecific neutralizing antibody responses were more likely to develop fever than children with cross-neutralizing responses. Preexisting DENV neutralizing antibodies are correlated with protection from dengue disease

Lack of Durable Cross-Neutralizing Antibodies Against Zika Virus from Dengue Virus Infection

Cross-reactive antibodies elicited by dengue virus (DENV) infection might affect Zika virus infection and confound serologic tests. Recent data demonstrate neutralization of Zika virus by monoclonal antibodies or human serum collected early after DENV infection. Whether this finding is true in late DENV convalescence (>6 months after infection) is unknown. We studied late convalescent serum samples from persons with prior DENV or Zika virus exposure. Despite extensive cross-reactivity in IgG binding, Zika virus neutralization was not observed among primary DENV infections. We observed low-frequency (23%) Zika virus cross-neutralization in repeat DENV infections. DENV-immune persons who had Zika virus as a secondary infection had distinct populations of antibodies that neutralized DENVs and Zika virus, as shown by DENV-reactive antibody depletion experiments. These data suggest that most DENV infections do not induce durable, high-level Zika virus cross-neutralizing antibodies. Zika virus–specific antibody populations develop after Zika virus infection irrespective of prior DENV immunity

Antibodies targeting dengue virus envelope domain III are not required for serotype-specific protection or prevention of enhancement in vivo

The envelope (E) protein of dengue virus (DENV) is composed of three domains (EDI, EDII, EDIII) and is the main target of neutralizing antibodies. Many monoclonal antibodies that bind EDIII strongly neutralize DENV. However in vitro studies indicate that anti-EDIII antibodies contribute little to the neutralizing potency of human DENV-immune serum. In this study, we assess the role of anti-EDIII antibodies in mouse and human DENV-immune serum in neutralizing or enhancing DENV infection in mice. We demonstrate that EDIII-depleted human DENV-immune serum was protective against homologous DENV infection in vivo. Although EDIII-depleted DENV-immune mouse serum demonstrated decreased neutralization potency in vitro, reduced protection in some organs, and enhanced disease in vivo, administration of increased volumes of EDIII-depleted serum abrogated these effects. These data indicate that anti-EDIII antibodies contribute to protection and minimize enhancement when present, but can be replaced by neutralizing antibodies targeting other epitopes on the dengue virion

Unsuspected Dengue as a Cause of Acute Febrile Illness in Children and Adults in Western Nicaragua

Dengue is an emerging infectious disease of global significance. Suspected dengue, especially in children in Nicaragua’s heavily-urbanized capital of Managua, has been well documented, but unsuspected dengue among children and adults with undifferentitated fever has not

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

Dengue Virus prM-Specific Human Monoclonal Antibodies with Virus Replication-Enhancing Properties Recognize a Single Immunodominant Antigenic Site

ABSTRACT The proposed antibody-dependent enhancement (ADE) mechanism for severe dengue virus (DENV) disease suggests that non-neutralizing serotype cross-reactive antibodies generated during a primary infection facilitate entry into Fc receptor bearing cells during secondary infection, resulting in enhanced viral replication and severe disease. One group of cross-reactive antibodies that contributes considerably to this serum profile target the premembrane (prM) protein. We report here the isolation of a large panel of naturally occurring human monoclonal antibodies (MAbs) obtained from subjects following primary DENV serotype 1, 2, or 3 or secondary natural DENV infections or following primary DENV serotype 1 live attenuated virus vaccination to determine the antigenic landscape on the prM protein that is recognized by human antibodies. We isolated 25 prM-reactive human MAbs, encoded by diverse antibody-variable genes. Competition-binding studies revealed that all of the antibodies bound to a single major antigenic site on prM. Alanine scanning-based shotgun mutagenesis epitope mapping studies revealed diverse patterns of fine specificity of various clones, suggesting that different antibodies use varied binding poses to recognize several overlapping epitopes within the immunodominant site. Several of the antibodies interacted with epitopes on both prM and E protein residues. Despite the diverse genetic origins of the antibodies and differences in the fine specificity of their epitopes, each of these prM-reactive antibodies was capable of enhancing the DENV infection of Fc receptor-bearing cells. IMPORTANCE Antibodies may play a critical role in the pathogenesis of enhanced DENV infection and disease during secondary infections. A substantial proportion of enhancing antibodies generated in response to natural dengue infection are directed toward the prM protein. The fine specificity of human prM antibodies is not understood. Here, we isolated a panel of dengue prM-specific human monoclonal antibodies from individuals after infection in order to define the mode of molecular recognition by enhancing antibodies. We found that only a single antibody molecule can be bound to each prM protein at any given time. Distinct overlapping epitopes were mapped, but all of the epitopes lie within a single major antigenic site, suggesting that this antigenic domain forms an immunodominant region of the protein. Neutralization and antibody-dependent enhanced replication experiments showed that recognition of any of the epitopes within the major antigenic site on prM was sufficient to cause enhanced infection of target cells