Barbara Blacklaws

Infection of Dendritic Cells by the Maedi-Visna Lentivirus

ABSTRACT The early stages of lentivirus infection of dendritic cells have been studied in an in vivo model. Maedi-visna virus (MVV) is a natural pathogen of sheep with a tropism for macrophages, but the infection of dendritic cells has not been proven, largely because of the difficulties of definitively distinguishing the two cell types. Afferent lymphatic dendritic cells from sheep have been phenotypically characterized and separated from macrophages. Dendritic cells purified from experimentally infected sheep have been demonstrated not only to carry infectious MVV but also to be hosts of the virus themselves. The results of the in vivo infection experiments are supported by infections of purified afferent lymph dendritic cells in vitro, in which late reverse transcriptase products are demonstrated by PCR. The significance of the infection of afferent lymph dendritic cells is discussed in relation to the initial spread of lentivirus infection and the requirement for CD4 T cells.

Specificity of the Immune Response of Mice to Herpes Simplex Virus Glycoproteins B and D Constitutively Expressed on L Cell Lines

Mouse L cell lines have been developed which constitutively express glycoproteins B (gB) and D (gD) of herpes simplex virus type 1. When used to study the immune response of mice to the viral glycoproteins, it was found that both gB and gD induce a delayed type hypersensitivity response and both also induce an antibody response, but only the cell line expressing gD could stimulate the production of neutralizing antibody. Virus-specific cytotoxic T lymphocytes (CTLs) recognized gB expressed by the cell line and this line could also induce CTLs in mice. Recognition of gD by major histocompatibility complex class I restricted CTLs was never seen. Vaccination of mice with the cell lines provided protection from viral challenge and inhibited the establishment of a latent infection, although gD proved to be the better protective immunogen.

Small ruminant lentiviruses: Immunopathogenesis of visna-maedi and caprine arthritis and encephalitis virus

The small ruminant lentiviruses include the prototype for the genus, visna-maedi virus (VMV) as well as caprine arthritis encephalitis virus (CAEV). Infection of sheep or goats with these viruses causes slow, progressive, inflammatory pathology in many tissues, but the most common clinical signs result from pathology in the lung, mammary gland, central nervous system and joints. This review examines replication, immunity to and pathogenesis of these viruses and highlights major differences from and similarities to some of the other lentiviruses.

Immunogenicity of herpes simplex virus type 1 glycoproteins expressed in vaccinia virus recombinants

Vaccinia virus recombinants expressing glycoproteins B (vgB11), D (VgD52), E (gE/7.5 and gE/4B), G (gG-vac), H (gH-vac), and I (gI-vac) of HSV-1 were used to compare the protective response to these individual glycoproteins in the mouse. Glycoprotein D induced the best neutralizing antibody titers and the most increased rates of HSV clearance from the ear as well as good protection from the establishment of latent HSV infections in the sensory ganglia. Glycoprotein B also induced good neutralizing antibody titers and as great a protection from the establishment of latency as gD although the rate of virus clearance from the ear was not as great as after immunization with gD. Glycoprotein E induced weak neutralizing antibody but gG, gH, and gI did not show a neutralizing antibody response. At higher challenge doses of virus (10(6) PFU HSV-1 in the ear), gE induced a protective response by increasing the rate of virus clearance and reducing the acute infection of ganglia as compared to negative control immunized mice. However there was no protection from the establishment of latent infections after immunization with gE. No protective response was seen to gG, gH, or gl.

Expression of glycoprotein D of herpes simplex virus type 1 in a recombinant baculovirus: protective responses and T cell recognition of the recombinant-infected cell extracts

Recombinant baculoviruses expressing glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) have been generated. The proteins expressed from the recombinants have been characterized using monoclonal antibodies on Western blots and by immunoprecipitation. Partially glycosylated 48K polypeptides have been identified as products of the gD gene. Polyclonal sera from H-2k mice infected with HSV-1 recognized the same polypeptides. Furthermore, draining lymph node cells from H-2k mice infected with HSV-1 proliferated in vitro in response to recombinant-infected cell extracts. Immunization with such extracts generated high titre complement-dependent and -independent neutralizing antibody and the mice were protected against a challenge with HSV-1.

Induction of protective immunity with antibody to herpes simplex virus type 1 glycoprotein H (gH) and analysis of the immune response to gH expressed in recombinant vaccinia virus

Passive administration of neutralizing monoclonal antibody (MAb) to glycoprotein H (gH) of herpes simplex virus type 1 (HSV-1) was found to protect mice from an HSV-1 strain SC16 challenge infection. To investigate further the protective potential of gH, recombinant vaccinia viruses were constructed which expressed the HSV-1 gH open reading frame under the control of the vaccinia virus 7.5K early/late promoter or the 4b late promoter. Immunization with recombinant viruses, however, did not induce the production of neutralizing antisera and the mice were not protected from zosteriform spread or the establishment of latent infection following viral challenge. The gH produced by the recombinant vaccinia viruses differed in electrophoretic mobility and antigenicity from authentic HSV-1 gH. Only one of three neutralizing MAbs specific for conformational epitopes on gH was able to immunoprecipitate gH synthesized in recombinant vaccinia virus-infected cells. In addition cell surface expression of gH was not detected in cells infected with the recombinant vaccinia viruses.

Induction of antibody responses to African horse sickness virus (AHSV) in ponies after vaccination with recombinant modified vaccinia Ankara (MVA).

Background African horse sickness virus (AHSV) causes a non-contagious, infectious disease in equids, with mortality rates that can exceed 90% in susceptible horse populations. AHSV vaccines play a crucial role in the control of the disease; however, there are concerns over the use of polyvalent live attenuated vaccines particularly in areas where AHSV is not endemic. Therefore, it is important to consider alternative approaches for AHSV vaccine development. We have carried out a pilot study to investigate the ability of recombinant modified vaccinia Ankara (MVA) vaccines expressing VP2, VP7 or NS3 genes of AHSV to stimulate immune responses against AHSV antigens in the horse. Methodology/Principal Findings VP2, VP7 and NS3 genes from AHSV-4/Madrid87 were cloned into the vaccinia transfer vector pSC11 and recombinant MVA viruses generated. Antigen expression or transcription of the AHSV genes from cells infected with the recombinant viruses was confirmed. Pairs of ponies were vaccinated with MVAVP2, MVAVP7 or MVANS3 and both MVA vector and AHSV antigen-specific antibody responses were analysed. Vaccination with MVAVP2 induced a strong AHSV neutralising antibody response (VN titre up to a value of 2). MVAVP7 also induced AHSV antigen–specific responses, detected by western blotting. NS3 specific antibody responses were not detected. Conclusions This pilot study demonstrates the immunogenicity of recombinant MVA vectored AHSV vaccines, in particular MVAVP2, and indicates that further work to investigate whether these vaccines would confer protection from lethal AHSV challenge in the horse is justifiable.

Immunological memory to herpes simplex virus type 1 glycoproteins B and D in mice

Mouse L cell lines constitutively expressing glycoproteins B or D of herpes simplex virus type 1 (LTKgB and LTKgD respectively) were used to study the longevity of the immune response to these viral glycoproteins in mice. Two immunizations with the cell lines were necessary to induce a persisting antibody response (present for over 200 days). Only LTKgD induced a neutralizing at antibody response in mice and this also remained at high titres over 200 days after two inoculations. The presence in mice of precursor cytotoxic T lymphocytes specific for gB expressed in the L cells was also shown up to 270 days after immunization. Mice immunized with the cell lines showed an increased rate of virus clearance from the ear pinna, inoculation with LTKgD resulting in more clearance than LTKgB at 7 days post-immunization. This type of protection was reduced with time after inoculation, until by day 161 there was no significant difference in virus titres between immunized and control groups. However, LTKgD immunization protected against the establishment of latent infections in the ganglia of mice even up to 186 days post-inoculation.

Activation of murine dendritic cells and macrophages induced by Salmonella enterica serovar Typhimurium

Macrophages and dendritic cells (DCs) are antigen‐presenting cells (APCs), and the direct involvement of both cell types in the immune response to Salmonella has been identified. In this study we analysed the phenotypic and functional changes that take place in murine macrophages and DCs in response to live and heat‐killed Salmonella enterica serovar Typhimurium. Both types of cell secreted proinflammatory cytokines and nitric oxide (NO) in response to live and heat‐killed salmonellae. Bacterial stimulation also resulted in up‐regulation of costimulatory molecules on macrophages and DCs. The expression of major histocompatibility complex (MHC) class II molecules by macrophages and DCs was differentially regulated by interferon (IFN)‐γ and salmonellae. Live and heat‐killed salmonellae as well as lipopolysaccharide (LPS) inhibited the up‐regulation of MHC class II expression induced by IFN‐γ on macrophages but not on DCs. Macrophages as well as DCs presented Salmonella‐derived antigen to CD4 T cells, although DCs were much more efficient than macrophages at stimulating CD4 T‐cell cytokine release. Macrophages are effective in the uptake and killing of bacteria whilst DCs specialize in antigen presentation. This study showed that the viability of salmonellae was not essential for activation of APCs but, unlike live bacteria, prolonged contact with heat‐killed bacteria was necessary to obtain maximal expression of the activation markers studied.

Intestinal intraepithelial lymphocyte activation promotes innate antiviral resistance

Unrelenting environmental challenges to the gut epithelium place particular demands on the local immune system. In this context, intestinal intraepithelial lymphocytes (IEL) compose a large, highly conserved T cell compartment, hypothesized to provide a first line of defence via cytolysis of dysregulated intestinal epithelial cells (IEC) and cytokine-mediated re-growth of healthy IEC. Here we show that one of the most conspicuous impacts of activated IEL on IEC is the functional upregulation of antiviral interferon (IFN)-responsive genes, mediated by the collective actions of IFNs with other cytokines. Indeed, IEL activation in vivo rapidly provoked type I/III IFN receptor-dependent upregulation of IFN-responsive genes in the villus epithelium. Consistent with this, activated IEL mediators protected cells against virus infection in vitro, and pre-activation of IEL in vivo profoundly limited norovirus infection. Hence, intraepithelial T cell activation offers an overt means to promote the innate antiviral potential of the intestinal epithelium.