Induction of Type I Interferon Secretion through Recombinant Newcastle Disease Virus Expressing Measles Virus Hemagglutinin Stimulates Antibody Secretion in the Presence of Maternal Antibodies▿

Measles virus (MV) vaccine effectively protects seronegative individuals against infection. However, inhibition of vaccine-induced seroconversion by maternal antibodies after vaccination remains a problem, as it leaves infants susceptible to MV infection. In cotton rats, passive transfer of MV-specific IgG mimics maternal antibodies and inhibits vaccine-induced seroconversion. Here, we report that immunization in the presence of passively transferred IgG inhibits the secretion of neutralizing antibodies but not the generation of MV-specific B cells. This finding suggested that MV-specific B cells require an additional stimulus to mature into antibody-secreting plasma cells. In order to provide such a stimulus, we generated a recombinant Newcastle disease virus (NDV) expressing the MV hemagglutinin (NDV-H). In contrast to MV, NDV-H induced high levels of type I interferon in plasmacytoid dendritic cells and in lung tissue. In cotton rats immunized with NDV-H, neutralizing antibodies were also generated in the presence of passively transferred antibodies. In the latter case, however, the level and kinetics of antibody generation were reduced. In vitro, alpha interferon stimulated the activation of MV-specific B cells from MV-immune spleen cells. NDV infection (which induces alpha interferon) had the same effect, and stimulation could be abrogated by antibodies neutralizing alpha interferon, but not interleukin 6 (IL-6). In vivo, coapplication of UV-inactivated MV with NDV led to increased MV-specific antibody production in the presence and absence of passively transferred antibodies. These data indicate that MV-specific B cells are being generated after immunization in the presence of maternal antibodies and that the provision of alpha interferon as an additional signal leads to antibody secretion

Analysis of cotton rat CD150 expression and tissue distribution.

<p>The presence of cotton rat CD150 was determined on spleen cells and lymph node cells stimulated with Concanavalin A for 24 hours (B and D), and ex vivo leukocytes from spleen (A), thymus (C), Peyer’s patches (F) and lymph node (E) (anti-CD150 black line; grey area isotype control).</p

Predicted amino acid sequence alignment of cotton rat, human and mouse CD150.

<p>Spaces (indicated by dashes) denote gaps generated during alignment for optimal sequence comparison. Dark shading represents fully conserved residues, while light shading indicates conserved changes. The predicted signal peptide and transmembrane domain of human CD150 are underlined. Potential N-linked glycosylation sites are circled, four cystine residues predicted to form disulfide bonds are denoted by asterisks and three tyrosine-based switch motifs are boxed.</p

MV infection of CD150 expressing 293T cells.

<p>(A) HEK 293T and 293T-CrCD150 cells were infected with 50 pfu/well of wild type MV (Bilthoven strain) and plaques were counted 48 hours post-infection. The difference in the number of virus induced plaques was statistically significant between both cell types (p<0.001, ANOVA). (B) After infection of 293T-HuCD150 and 293T-CrCD150 with MV (Bilthoven strain) viral titers were measured at different time intervals. Infection with wild type MV (Bilthoven strain) caused the formation of numerous small viral plaques on 293T-CrCD150 cells (C) and large plaques on 293T-HuCD150 cells (D) (40x magnification).</p

Infectivity of VSV pseudotype viruses on 293T cells expressing cotton rat and human CD150.

<p>(A) HEK 293T cells were stably transfected with plasmids expressing cotton rat (293T-CrCD150) or human CD150 (293T-HuCD150). Expression of CD150 was verified with antibodies against the cotton rat or human CD150 molecule, respectively. (B) 293T, 293T-CrCD150 and 293T-HuCD150 cells were infected with VSVΔG*-Ed-H/F. The infectivity titer was calculated by counting the number of GFP expressing cells. There was a no statistically significant difference in titer between HEK 293T, 293T-CrCD150 and 293T-HuCD150 cells. (C) 293T, 293T-CrCD150 and 293T-HuCD150 cells were infected with VSVΔG*-WTF-H/F. The infectivity titer was calculated by counting the number of GFP expressing cells. There was a significant increase in titer between HEK 293T, 293T-CrCD150 and 293T-HuCD150 cells (p<0.001, ANOVA).</p

Cotton Rat (Sigmodon hispidus) Signaling Lymphocyte Activation Molecule (CD150) Is an Entry Receptor for Measles Virus

Cotton rats (Sigmodon hispidus) replicate measles virus (MV) after intranasal infection in the respiratory tract and lymphoid tissue. We have cloned the cotton rat signaling lymphocytic activation molecule (CD150, SLAM) in order to investigate its role as a potential receptor for MV. Cotton rat CD150 displays 58% and 78% amino acid homology with human and mouse CD150, respectively. By staining with a newly generated cotton rat CD150 specific monoclonal antibody expression of CD150 was confirmed in cotton rat lymphoid cells and in tissues with a pattern of expression similar to mouse and humans. Previously, binding of MV hemagglutinin has been shown to be dependent on amino acids 60, 61 and 63 in the V region of CD150. The human molecule contains isoleucine, histidine and valine at these positions and binds to MV-H whereas the mouse molecule contains valine, arginine and leucine and does not function as a receptor for MV. In the cotton rat molecule, amino acids 61 and 63 are identical with the mouse molecule and amino acid 60 with the human molecule. After transfection with cotton rat CD150 HEK 293 T cells became susceptible to infection with single cycle VSV pseudotype virus expressing wild type MV glycoproteins and with a MV wildtype virus. After infection, cells expressing cotton rat CD150 replicated virus to lower levels than cells expressing the human molecule and formed smaller plaques. These data might explain why the cotton rat is a semipermissive model for measles virus infection