Although often considered to be ineffective against intracellular bacteria, Abs, in the absence of lymphocytes, have been shown previously to protect SCID mice from lethal infection by the obligate intracellular bacterium Ehrlichia chaffeensis, even when administered well after infection has been established. To identify characteristics of Abs that are critical for host defense during this intracellular infection, a panel of Ehrlichia-specific mAbs was generated and analyzed. Among 100 Abs recovered, 39 recognized an amino-terminal hypervariable region of an outer membrane protein (OMP), demonstrating that the OMPs are both antigenically variable and immunodominant. A subset of 16 representative OMP-specific Abs was further examined to identify characteristics that were essential for in vivo efficacy. The highly effective Abs recognized a linear epitope within the first hypervariable region of OMP-1g. Only IgG were found to be effective, and among the effective IgG, the following hierarchy was observed: IgG2a > IgG3 = IgG2b. The most striking characteristics of the highly effective Abs were their picomolar binding affinities and long binding t1/2. Thus, although epitope recognition and isotype use may contribute to efficacy, high affinity may be a critical characteristic of Abs that can act effectively during this intracellular bacterial infection.

Ehrlichia chaffeensis, an obligate intracellular, tick-transmitted bacterium, is susceptible to antibody-mediated host defense, but the mechanism by which this occurs is not understood. One possible explanation is that antibodies directly access the bacteria in the extracellular environment of the host, perhaps during bacterial intercellular transfer. Accordingly, we investigated whether bacteria could be found outside of host cells during infection. Host cell-free plasma obtained from infected mice was found to contain ehrlichiae, and the host cell-free ehrlichiae readily transferred disease to susceptible SCID recipients. The host cell-free ehrlichiae were found during infection of both immunocompetent BALB/c and immunocompromised BALB/c-scid mice and reached levels as high as 10 8 /ml in plasma during persistent infection in SCID mice. Approximately 10% of the blood-borne bacteria were found outside of host cells. Although it is generally accepted that replication of ehrlichiae occurs only within host cells, the cell-free bacteria were shown to undergo DNA replication and cell division in vitro for 3 to 5 days when incubated at 37°C in plasma. Paradoxically, both infectivity and virulence were lost after 24 h of ex vivo culture. The data indicate that E. chaffeensis is exposed to the extracellular milieu during infection, presumably during intercellular transfer, and reveal that these intracellular bacteria do not require the environment of the host cell for replication. Our findings reveal a possible mechanism by which antibodies can access the intracellular bacteria upon their release into the extracellular milieu and mediate host defense and also have implications for understanding the replication and transmission of this vectorborne pathogen.

Previous studies of Ehrlichia chaffeensis infection in the mouse have demonstrated that passive transfer of polyclonal Abs from resistant immunocompetent mice to susceptible SCID mice ameliorated infection and disease, even when Abs were administered during established infection. To identify particular Abs that could mediate bacterial clearance in vivo, E. chaffeensis-specific mAbs were generated and administered to infected SCID mice. Bacterial infection in the livers was significantly lowered after administration of either of two Abs of different isotypes (IgG2a and IgG3). Moreover, repeated administration of one Ab (Ec56.5; IgG2a) rescued mice from an otherwise lethal infection for at least 5 wk. Both protective Abs recognized the E. chaffeensis major outer membrane protein (OMP)-1g. Further studies revealed that both Abs recognized closely related epitopes within the amino terminus of the first hypervariable region of OMP-1g. Analyses of human sera showed that E. chaffeensis-infected patients also generated serological responses to OMP-1g hypervariable region 1, indicating that humans and mice recognize identical or closely related epitopes. These studies demonstrate that OMP-specific mAbs can mediate bacterial elimination in SCID mice, and indicate that Abs, in the absence of cell-mediated immunity, can play a significant role in host defense during infection by this obligate intracellular bacterium.

Our laboratory has been investigating the mechanisms of host defense during Ehrlichia chaffeensis infection in the mouse. Although major roles are clearly played by T cells, we found that antibodies could also control infection in both normal and immunocompromised SCID mice, and could protect the latter from lethal infection. Antibodies are not generally effective during such intracellular infections, so we would like to understand exactly how antibodies can mediate immunity in this model. We have found that much of the humoral immune response is directed at the bacterial outer membrane proteins (OMPs), and that highly effective OMP antibodies (mostly IgG2a) exhibited picomolar affinities and very long binding half-lives. These antibodies, which could mediate bacterial clearance from tissues as early as 24 hours after administration, require host Fc receptors for their function(s). In contrast, we have failed to find any role for complement or reactive nitrogen intermediates, or for neutrophils, or for NK cells. One possible mechanism is that antibodies or immune complexes trigger microbiocidal activities in infected macrophages that lead to the elimination of bacteria residing inside host macrophages. Alternatively, it is proposed that antibodies opsonize bacteria exposed during intercellular transfer. This notion is supported by studies that have demonstrated the presence of bacteria in the extracellular milieu during infection, and suggests that our understanding of the behavior of the bacterium in the host may be key to our understanding of its susceptibility to antibody-mediated host defenses.