Immunological responses and viral modulatory effects of vaccination with recombinant modified vaccinia virus Ankara (rMVA) expressing structural and regulatory transgenes of simian immunodeficiency virus (SIVmac32H/J5M).

BACKGROUND: The immunogenicity and protective efficacy of recombinant modified vaccinia virus Ankara (rMVA) vectors expressing structural (gag/pol, env) and regulatory (tat, rev, nef) genes of SIVmac251/32H-J5 (rMVA-J5) were assessed. METHODS: Immunization with rMVA constructs (2.5 x 10(7) IU) 32, 20 and 8 weeks pre-challenge was compared with 32 and 20 weeks but with a final boost 8 weeks pre-challenge with 2 x 10(6) fixed-inactivated HSC-F4 cells infected with SIVmac32H. Controls received rMVA vectors expressing an irrelevant transgene or were naïve challenge controls. All received 10 MID(50) SIVmac32H/J5 intravenously. RESULTS: Vaccinates immunized with rMVA-J5 exhibited significant, albeit transient, control of peak primary viraemia despite inconsistent and variable immune responses elicted by vaccination. Humoral and cellular responses to Env were most consistent, with lower responses to Nef, Rev and Tat. Increasing titres of anti-vaccinia neutralizing antibodies reflected the number and dose of rMVA inoculations. CONCLUSIONS: Improved combinations of viral vectors are required to elicit appropriate immune responses to control viral replication.

Preparation and characterization of new challenge stocks of SIVmac32H J5 following rapid serial passage of virus in vivo.

BACKGROUND: A new challenge stock of the simian immunodeficiency virus SIVmacJ5 has been produced following passage in vivo. METHODS: SIVmacJ5 3/92 (J5M), was passaged serially through cynomolgus macaques (Macaca fascicularis) by intravenous inoculation of infected spleen cells isolated and prepared 14 days post-infection. Two challenge stocks, SIVmacJ5 S61MLN and SIVmacJ5 S62spl, were prepared by culture of lymphoid tissue ex vivo. RESULTS: These virus stocks appeared better adapted for replication in M. fascicularis as demonstrated by a greater persistence of recoverable live virus from the periphery and increased pathology in lymphoid tissues 20 weeks post-challenge as detected by immunohistochemistry. Sequence analysis of the envelope gene from these stocks did not identify marked diversification of sequence as a result of this procedure. CONCLUSIONS: These stocks display more robust peripheral persistence and tissue pathology in cynomolgus macaques and should prove valuable analysing recombinant vaccines based upon SIVmacJ5 transgenes.

Mechanisms of loss of functional dendritic cells in HIV-1 infection.

Dendritic cells (DC) are lost from blood and skin during injection with HIV-1; those remaining show a reduced capacity to stimulate T cell proliferation [S. C. Knight, AIDS 10, 807-817]. Our recent studies investigate mechanisms underlying these effects. DC exposed to HIV-1 vitro can act as targets for cytotoxic T cells, although optimal killing was not obtained until DC were exposed to HIV-1 for 3 days. This cytotoxicity may provide a feedback mechanism by which DC that have presented antigens are removed. However, this effect could also contribute to the reduction in DC during persistent infection. We have also investigated the effect of exposure to HIV-1 on DC function. DC exposed to HIV-1 IIIB virus for 2 h stimulated primary proliferative and cytotoxic T cell responses in vitro; these effects may be similar to those occurring during the early activation of protective antiviral immunity in vivo. After exposure of DC to virus for 5 days, stimulation of allogeneic T cells was reduced. However, a different situation applied when using DC developed from CD34+ cord blood stem cells under the influence of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor alpha that were exposed at 24 h to the same virus. These DC showed low levels of infection similar to peripheral blood DC but in contrast stimulated normal allogeneic T cell proliferation. The capacity of DC exposed to HIV-1 to stimulate T cell proliferation or to show a blocked stimulatory capacity may thus depend not only on the length of the exposure to virus but also on the maturational state of the DC. Loss in DC numbers and function on exposure to HIV-1 may result in lower levels of stimulation of T cells, which in turn may be instrumental in reduction of T cell numbers.

Antigen-presenting cells but not lymphocytes in the joint may indicate the cause of reactive arthritis.

T cells and antigen-presenting cells (APC) accumulate in the joint in reactive arthritis and there are reports that the T cells are a population selected for responsiveness to the causative agent. In this work, the latter view is questioned by detailed studies of the antigen specificities of the lymphocytes within the joint (SFMC) and peripheral blood (PBMC) of patients with reactive arthritis triggered by infection with Chlamydia trachomatis. Using a hanging-drop microculture system. SFMC displayed enhanced responses not only to antigens from the triggering organism, but also to other antigens, including PPD and tetanus toxoid, to which the patients were likely to have had prior exposure. No evidence was obtained for a dominant cross-reactive T-cell response to epitopes common to these antigen preparations, confirming the polyclonal nature of the infiltrate. In contrast to the broad specificity of the T-cell infiltrate, two experimental approaches indicated that APC within the joint carried chlamydial antigen. The failure of antigen-bearing APC to interact with T cells at this site may underlie the inability to clear microbial antigen from the joint.

Murine retrovirus induces defects in the function of dendritic cells at early stages of infection.

The infection and function of lymph node dendritic cells (DC) were analyzed at different time points of Rauscher leukemia virus infection in mice (3, 7, 14, and 21 days). Infection of DC was apparent after 3 days and significant infection (1-10% of the DC population) was documented after 7 days. DC from infected mice as early as 3 days postinfection had a reduced ability to stimulate allogeneic normal T cells in the mixed lymphocyte reaction. T cells did become infected during the coculture but block of cross-infection of T cells by zidovudine did not abolish the inhibitory effect. Other DC-dependent responses were also reduced on infection including DC-stimulated responses to influenza virus. ConA and PMA induced an increase in [Ca2+]i level in DC from control mice. A low baseline level of [Ca2+]i in DC from infected mice and reduced calcium mobilization upon ConA stimulation was found at all periods of infection. Ultraviolet-inactivated Rauscher leukemia virus failed to provoke significant changes in DC function in vivo. Six or 7 days after RLV infection DC expressed lower levels of Iad but not H2Dd molecules in parallel with lower expression of some adhesion molecules (CD18, CD54, CD44). No differences in expression of B7 surface antigen between control and infected mice were obtained. We did not find any evidence for the induction of apoptosis of naive syngeneic or allogeneic T cells by infected dendritic cells. The changes in DC function may have implications for the pathogenesis of retroviral infections including HIV infection.

Primary human T-cell responses to the major outer membrane protein of Chlamydia trachomatis.

The major outer membrane protein (MOMP) of Chlamydia trachomatis is the main candidate antigen for a synthetic vaccine against chlamydial infection. Antibodies to surface-exposed epitopes on MOMP neutralize chlamydial infectivity but little is known about T-cell recognition of the molecule. We have measured primary human T-cell responses to recombinant fragments of MOMP as well as to the whole organism and synthetic MOMP peptides. Using antigen-pulsed low density cells (LDC) we were able to stimulate proliferative responses with T cells from most naive individuals. This response was antigen dose dependent and displayed an absolute requirement for dendritic cells in the antigen-presenting cell (APC) population. Several T-cell epitopes were identified in MOMP and one which stimulated T cells from 80% of donors was resolved as a 12 amino acid synthetic peptide. Dual cell surface labelling and cell cycle analysis by FACS revealed that both CD4+ and CD8+ T cells were stimulated in these cultures. The fact that we were able to obtain proliferative responses and interferon-gamma (IFN-gamma) production to MOMP using cells from cord bloods confirmed that these are genuine primary responses. These experiments have identified a region on MOMP, to which T cells from most humans make a primary response, which may be useful in a chlamydial vaccine. The approach is useful for vaccine development in general.