Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein.

Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens.

Recombinant parainfluenza virus 5 (PIV5) expressing the influenza A virus hemagglutinin provides immunity in mice to influenza A virus challenge.

Parainfluenza virus type 5 (PIV5), formerly known as simian virus 5 (SV5), is a non-segmented negative strand RNA virus that offers several advantages as a vaccine vector. PIV5 infects many cell types causing little cytopathic effect, it replicates in the cytoplasm of infected cells, and does not have a DNA phase in its life cycle thus avoiding the possibility of introducing foreign genes into the host DNA genome. Importantly, PIV5 can infect humans but it is not associated with any known human illness. PIV5 grows well in tissue culture cells, including Vero cells, which have been approved for vaccine production, and the virus can be obtained easily from the media. To test the feasibility of using PIV5 as a live vaccine vector, the hemagglutinin (HA) gene from influenza A virus strain A/Udorn/72 (H3N2) was inserted into the PIV5 genome as an extra gene between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Recombinant PIV5 containing the HA gene of Udorn (rPIV5-H3) was recovered and it replicated similarly to wild type PIV5, both in vitro and in vivo. The HA protein expressed by rPIV5-H3-infected cells was incorporated into the virions and addition of the HA gene did not increase virus virulence in mice. The efficacy of rPIV5-H3 as a live vaccine was examined in 6-week-old BALB/c mice. The results show that a single dose inoculation provides broad and considerable immunity against influenza A virus infection.

Social experience alters the response to social stress in mice.

Individual differences in the response to stressful stimuli have been documented in humans and in a variety of animal species. Recently, we demonstrated that social stress induced a state of glucocorticoid (GC) resistance in mouse splenocytes, however this response was highly variable among cage mates. Since these studies were conducted using inbred mice (C57BL/6), it was suggested that environmental factors were the source of this variability. The following study examined possible factors that may have contributed to the development of individual differences in the susceptibility of mice to social stress. First, the effect of rearing conditions was studied by comparing the development of GC resistance in mice reared in isolation or in groups. In addition, the effect of previous social experiences was studied in mice that were re-housed to facilitate the formation of new social hierarchies in the cages. The results indicated that isolation altered the behavior of the mice during the social stress, but did not affect the development of GC resistance in response to the stress. Re-housing and the resulting loss of social status increased the susceptibility of mice to the development of GC resistance following social stress. Together, these findings indicate that environmental factors, such as previous social experiences, may alter the susceptibility to the effects of future social stress in inbred mice.

Expression of glucocorticoid resistance following social stress requires a second signal.

Stimulation of splenocytes from socially stressed mice [social disruption (SDR)] with Gram-negative bacterial lipopolysaccharide (LPS) revealed a state of functional glucocorticoid (GC) resistance. LPS-stimulated splenocytes were less sensitive to the inhibitory effects of corticosterone. This study demonstrated that activation signals were required for the expression of splenic GC resistance. The results demonstrated that six cycles of SDR induced splenomegaly and increased the number of CD11b-positive monocytes. SDR also increased the viability of cultured, nonstimulated splenocytes, and addition of corticosterone reduced the viability of these cells in a dose-dependent manner. However, following stimulation with LPS, the sensitivity of SDR splenocytes to GC was reduced. Similar results were obtained using lipid A, a fraction of the LPS molecule that binds to Toll-like receptor (TLR)4. Furthermore, C3H/HeJ mice that do not possess a functional TLR4 molecule responded to SDR with an increased number of CD11b-positive monocytes in the spleen and increased viability of nonstimulated splenocytes. However, neither LPS nor lipid A stimulation resulted in the expression of GC resistance. Together, these findings suggest that the expression of GC resistance in response to SDR requires a second signal that can be provided by ligation of TLR4.