Previous studies compared uptake by dendritic cells (DC) of 20, 40, 100, 200, 500, 1000, and 2000 nm beads in vivo. When beads were used as antigen carriers, bead size influenced antibody responses and induction of IFN-gamma-producing CD4 and CD8 T cells. Beads of 40-50 nm were taken up preferentially by DC and induced particularly strong immunity. Herein, we examine immunity induced by minute differences in nanobead size, specifically within a narrow viral-sized range (20, 40, 49, 67, 93, 101, and 123 nm), to see if bead carrier size influenced the induction of type 1 or type 2 cells as demonstrated by the production of IFN-gamma or IL-4. In vivo uptake by DC was assessed for selected sizes in this range. Responses to whole ovalbumin (OVA) or the OVA-derived CD8 T cell peptide epitope (SIINFEKL) were tested. After one immunization with beads-OVA, IFN-gamma responses to both OVA and SIINFEKL were significantly better with 40 and 49 nm beads than other sizes, while, in contrast, IL-4 responses to OVA were higher after immunization with OVA conjugated to larger beads (93, 101, and 123 nm). Thus IFN-gamma induction from CD8 T cells was limited to 40-49 nm beads, while CD4 T cell activation and IL-4 were induced by 93-123 nm beads-OVA. After two immunizations, there were comparable high levels of IFN-gamma produced with 40 and 49 beads and IL-4 reactivity was still higher for larger beads (93, 101, 123 nm). Production of IgG1 was seen across the full range of bead sizes, increasing after two immunizations. Since protection against respiratory syncytial virus (RSV) depends on strong IFN responses, while IL-4 responses are reported to cause asthma-like symptoms, immunization with RSV antigens on the 49 nm carrier beads could provide the basis for a suitable vaccine. When the 49 nm beads were conjugated to RSV proteins G88 (surface) or M2.1 (internal capsid), one immunization with G88 induced high levels of IFN-gamma and low levels of IL-4. IL-4 increased with two immunizations. Beads-M2.1 induced only moderate levels of IFN-gamma and low titer antibody after two immunizations. Mice vaccinated once with G88-conjugated 49 nm beads and challenged intranasally with RSV strain A2 subtype showed reduced viral titers and recovered from weight loss more rapidly than mice immunized with M2.1-conjugated 49 nm beads or naive control mice. These results show that precise selection of nanobead size for vaccination can influence the type 1/type 2 cytokine balance after one immunization, and this will be useful in the development of effective vaccines against common human pathogens such as RSV.

Little is known about the functions of the matrix (M) protein of respiratory syncytial virus (RSV). By analogy with other negative-strand RNA viruses, the M protein should inhibit the viral polymerase prior to packaging and facilitate virion assembly. In this study, localization of the RSV M protein in infected cells and its association with the RSV nucleocapsid complex was investigated. RSV-infected cells were shown to contain characteristic cytoplasmic inclusions. Further analysis showed that these inclusions were localization sites of the M protein as well as the N, P, L and M2-1 proteins described previously. The M protein co-purified with viral ribonucleoproteins (RNPs) from RSVinfected cells. The transcriptase activity of purified RNPs was enhanced by treatment with antibodies to the M protein in a dose-dependent manner. These data suggest that the M protein is associated with RSV nucleocapsids and, like the matrix proteins of other negative-strand RNA viruses, can inhibit virus transcription.Human respiratory syncytial virus (RSV) is a non-segmented negative-strand RNA virus belonging to the family Paramyxoviridae, genus Pneumovirus (Collins et al., 1996). RSVinfected cells contain characteristic cytoplasmic inclusions. Previous studies have shown that these inclusions contain the nucleocapsid (N) protein, the viral polymerase (P and L proteins) (Collins et al., 1996) and the transcription elongation factor protein M2-1 (Garcia et al., 1993). It has been proposed that, as with Sendai virus, the P protein interacts with the newly synthesized N protein to prevent incorrect assembly of the nucleocapsid and to deliver it to the nascent RNA during genome replication (Curran et al., 1995 ;Garcia-Barreno et al., 1996), whereas the M2-1 protein is required for the synthesis of full-length viral RNA (Collins et al., 1996). These obser-

Replication complexes are membrane-bound cytoplasmic vacuoles involved in rubella virus (RV) replication. These structures can be identified by their characteristic morphology at the electron microscopy (EM) level and by their association with double-stranded (ds) RNA in immunogold labeling EM studies. Although these virus-induced structures bear some resemblance to lysosomes, their exact nature and origin are unknown. In this study, the localization of two lysosomal markers, lysosomal-associated membrane protein (Lamp-1) and acid phosphatase, relative to the replication complexes was examined by light and electron microscopy. Confocal microscopy using antibodies to dsRNA and Lamp-1 showed colocalization of these two markers in the cytoplasm of RV-infected cells. Immunogold labeling EM studies using antibodies to Lamp-1 confirmed that Lamp-1 was associated with RV replication complexes. EM histochemical studies demonstrated the presence of acid phosphatase in the vacuoles of RV replication complexes. Taken together, these studies show that RV replication complexes are virus-modified lysosomes.

Cytoplasmic inclusions in respiratory syncytial virus-infected cells comprising viral nucleocapsid proteins (L,N Respiratory syncytial virus (RSV), a member of the familyParamyxoviridae, is one of the most important viral agents causing lower respiratory tract disease in infants, the elderly, and immunocompromised patients of all ages (3, 6, 21). The genome of RSV is a nonsegmented negative-strand RNA encoding 11 proteins (3). In RSV-infected cells, the viral RNA with the L (polymerase), N (nucleocapsid), P (phosphoprotein), and M2-1 proteins form the polymerase complex in which the transcription of messenger and genomic RNA takes place. As the cytoplasmic inclusions in RSV-infected cells have been shown to contain all elements of the polymerase complex and are capable of transcription in isolation (1, 7), it is presumed that they are major sites of viral transcription.Several studies have shown that the N protein is the major driver for the formation of these cytoplasmic inclusions. N associates with viral RNA, and N-RNA complexes are resistant to RNase treatment (18). Inclusion-like structures are formed when the N and P proteins are coexpressed in cells (7), and this association results from a specific protein-protein interaction between N and P, which can be disrupted by mutagenesis (8, 24). Garcia et al. (7) also showed that the M2-1 protein is present in cytoplasmic inclusions; subsequent investigations confirmed that the association of the M2-1 protein with inclusions resulted from its association with P (16).We previously reported that the RSV M protein is also found in cytoplasmic inclusions late during infection, in association with the N, P, and M2-1 proteins (11). Since we have also shown that the M protein inhibits virus transcription (11), the role of the M protein in cytoplasmic inclusions may be to inhibit viral transcription as a prelude to viral assembly and budding, driven by the M protein bringing cytoplasmic nucleocapsids into association with RSV envelope proteins (10). The concept is supported by data indicating specific interactions between M and the cytoplasmic domains of envelope glycoproteins (10).To date, it is not known how M becomes associated with the nucleocapsid complex. In the current study, we demonstrate that the N terminus of M can bind directly to M2-1 in a cell-free assay and that M colocalizes with M2-1 in the cytoplasm of cells either infected with RSV or expressing only M and M2-1 proteins. Using a cotransfection system, it was demonstrated that M associates with inclusion-like structures formed by N and P only in the presence of M2-1. MATERIALS AND METHODS Cells and virus.Human epithelial (HEp2) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS) at 37°C and 5% CO 2 . RSV subgroup A strain A2 (a gift from Paul Young, University of Queensland, Brisbane, Australia) was grown in HEp2 cells as previously described (9). To prepare the virus stock, an 80% confluent cell monolayer was infected with RSV at a multiplicity of infect...