Mike Dennis

Protective mucosal immunity elicited by targeted iliac lymph node immunization with a subunit SIV envelope and core vaccine in macaques.

Prevention of sexually transmitted HIV infection was investigated in macaques by immunization with a recombinant SIV (simian immunodeficiency virus) envelope gp120 and core p27 vaccine. In two independent series of experiments, we used the novel targeted iliac lymph node (TILN) route of immunization, aiming close to the iliac lymph nodes draining the genitorectal mucosa. Rectal challenge with the SIVmac 32H J5 molecular clone in two series induced total protection in four out of seven macaques immunized by TILN, compared with infection in 13 of 14 unimmunized macaques or immunized by other routes (P = 0.025). The remaining three macaques showed either a decrease in viral load (>90%) or transient viremia, indicating that all seven TILN–immunized macaques showed total or partial protection (P = 0.001). Protection was associated with significant increase in the iliac lymph nodes of lgA antibody–secreting cells to p27 (P < 0.02), CD8–suppressor factor (P< 0.01), and the chemokines RANTES and MIP–1β (P< 0.01).

The role of γ δ T cells in generating antiviral factors and β‐chemokines in protection against mucosal simian immunodeficiency virus infection

In view of the role of γ δ+ T cells in mucosal protection against infection, the proportion of γ δ T cells was examined in cells eluted from lymphoid and mucosal tissues of macaques immunized with simian immunodeficiency virus (SIV) gp120 and p27 in alum and challenged with live SIV by the rectal mucosal route. This revealed a significant increase in γ δ T cells eluted from the rectal mucosa (p < 0.01) and the related iliac lymph nodes (p < 0.0001) in protected as compared with infected macaques. Preferential homing of PKH‐26‐labeled γ δ+ T cells from the primed iliac lymph nodes to the rectal and cervico‐vaginal mucosa was demonstrated after targeted iliac lymph node as compared with i. m. immunization. Investigations of the mechanism of protection revealed that γ δ+ T cells can generate antiviral factors, RANTES, macrophage inflammatory protein (MIP)‐1α and MIP‐1β which can prevent SIV infection by binding to the CCR5 coreceptors. Up‐regulation of γ δ+ T cells was demonstrated by immunization of macaques with heat shock protein (HSP)70 linked to peptides and with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). This was confirmed by in vitro studies showing that GM‐CSF can up‐regulate γ δ+ T cells from macaques immunized with HSP‐linked peptides but not those from naive animals. We suggest that a novel strategy of immunization with HSP70 linked to antigen may generate both cognate immunity to the antigen and innate immunity by virtue of up‐regulation of γ δ+ T cells. These cells generate antiviral factors and the three β‐chemokines that prevent binding and transmission of SIV or M‐tropic HIV by the CCR5 coreceptor.

Induction of simian immunodeficiency virus (SIV)-specific CTL in rhesus macaques by vaccination with modified vaccinia virus Ankara expressing SIV transgenes: influence of pre-existing anti-vector immunity.

A major aim in AIDS vaccine development is the definition of strategies to stimulate strong and durable cytotoxic T lymphocyte (CTL) responses. Here we report that simian immunodeficiency virus (SIV)-specific CTL developed in 4/4 macaques following a single intramuscular injection of modified vaccinia virus Ankara (MVA) constructs expressing both structural and regulatory/accessory genes of SIV. In two animals Nef-specific responses persisted, but other responses diminished and new responses were not revealed, following further vaccination. Vaccination of another two macaques, expressing Mamu A*01 MHC class I, with MVA constructs containing nef and gag-pol under the control of the moderate strength natural vaccinia virus early/late promoter P7.5, again induced an early Nef-specific response, whereas responses to Gag remained undetectable. Anti-vector immunity induced by this immunization was shown to prevent the efficient stimulation of CTL directed to the cognate Gag epitope, p11C C-M, following vaccination with another MVA construct expressing SIV Gag-Pol under a strong synthetic vaccinia virus-specific promoter. In contrast, vaccination of a previously unexposed animal resulted in a SIV-specific CTL response widely disseminated in lymphoid tissues including lymph nodes associated with the rectal and genital routes of SIV entry. Thus, despite the highly attenuated nature of MVA, repeated immunization may elicit sufficient anti-vector immunity to limit the effectiveness of later vaccination.

Up-regulation of beta-chemokines and down-modulation of CCR5 co-receptors inhibit simian immunodeficiency virus transmission in non-human primates.

A non‐cognate mechanism of protection against human immunodeficiency virus‐1 (HIV‐1) infection involves up‐regulation of β‐chemokines, which bind and may down‐modulate the CCR5 co‐receptors, thereby preventing transmission of M‐tropic HIV‐1. The objective of this investigation was to evaluate this mechanism in vivo in non‐human primates. Rhesus macaques were immunized by a modified targeted lymph nodes (TLN) route with recombinant simian immunodeficiency virus (SIV) glycoprotein 120 (gp120) and p27 in alum, and adsorbed recombinant granulocyte–macrophage colony‐stimulating factor (GM‐CSF) with either interleukin (IL)‐2 or IL‐4. Immunization induced significant increases in the concentrations of CD8 cell‐derived suppressor factor (CD8‐SF), regulated on activation normal T cells expressed and secreted (RANTES), macrophage inflammatory protein (MIP)‐1α and MIP‐1β, and down‐modulation of the proportion of cells expressing CCR5 (r = 0·737, P < 0·05). The macaques were then challenged with SIVmac 220 by the rectal mucosal route. The plasma SIVmac RNA showed a significant inverse correlation with the CD8‐SF or the concentration of the three β‐chemokines (r = 0·831 and 0·824, P < 0·01), but a positive correlation between the proportion of CCR5+ cells and SIVmac RNA (r = 0·613, P = 0·05). These results demonstrate for the first time in vivo that immunization up‐regulates β‐chemokines, which may down‐modulate CCR5 co‐receptors, and both functions are significantly correlated with the viral load. Hence, the non‐cognate β‐chemokine–CCR5 mechanism should be considered as complementary to specific immunity in vaccination against HIV.

Antibody-secreting cells specific for simian immunodeficiency virus antigens in lymphoid and mucosal tissues of immunized macaques.

Objectives:To examine whether the route of immunization affects the induction of antibody-secreting cells (ASC) in the circulation of macaques. The distribution of ASC in the rectal mucosa and lymphoid tissues following challenge with simian immunodeficiency virus (SIV) was investigated. Design:Macaques were immunized with recombinant SIV gp120 and p27 antigens by the targeted iliac lymph node (TILN) route of immunization or the nasal and rectal route, augmented by intramuscular immunization [naso-rectal intramuscular (NRI)]. The macaques were challenged with live SIV by the rectal route and ASC were assayed in the circulation before and after SIV challenge, and in the tissues removed at post-mortem. Methods:ASC were examined in the circulation by Elispot assay. Mononuclear cells were prepared from peripheral blood, iliac and axillary lymph nodes and spleen. Rectal tissue was treated by enzyme digestion to elute mononuclear cells. Results:TILN and NRI immunization induced circulating IgA and IgG ASC to both gp120 and p27. Following rectal challenge with SIV, TILN macaques were protected from infection whereas NRI route-immunized and unimmunized controls became infected. IgA ASC to p27 were increased significantly in the iliac lymph nodes of the TILN immunized macaques compared with unimmunized controls (P < 0.05). Only IgA ASC were found in the rectal mucosa of the immunized protected macaques but both IgA and IgG ASC were detected in the unimmunized infected macaques. Overall the number of IgG ASC specific for p27 was significantly higher in the infected NRI and control macaques than in the protected macaques (P < 0.02). A progressive increase in IgG but not IgA ASC was detected in the peripheral blood mononuclear cells of the unimmunized infected macaques. Conclusions:The results suggest that cells secreting IgA antibodies to p27 in the iliac lymph nodes of the TILN immunized macaques correlate significantly with protection from infection. The unimmunized infected macaques showed a progressive increase in IgG ASC in the peripheral blood after SIV challenge; this was found in the iliac and axillary lymph nodes and also in the spleen, suggesting that it is an immune response to the SIV infection.

The influence of humidity on the protective performance of a membrane based on poly(vinyl alcohol)

A solid-state NMR study has been used to rationalize the way humidity affects the barrier properties of a polyvinyl alcohol-based membrane. Polymer blends composed of polyvinyl alcohol–polyethyleneimine (PVOH–PEI) and polyvinyl alcohol–polydiallyldimethyl ammonium chloride (PVOH–PDADMAC) were investigated. At 90% relative humidity the PVOH–PEI material retains a PVOH-rich component that is detected in a cross-polarization NMR experiment and is therefore assumed to be relatively immobile. The barrier properties of this material are also largely retained at this relative humidity. Under the same conditions of humidity no signal is detected in the cross-polarization NMR experiment for the PVOH–PDADMAC system and the barrier properties of the PVOH–PDADMAC system are compromised. The persistence of the more rigid domains in the PVOH–PEI material is used to explain the barrier properties of this blend at high relative humidity. For the PVOH–PDADMAC system the cross-polarization signal is recovered and the barrier properties of the blend are restored to their original level as the humidity is lowered.

Analysis of SIV-specific CTL in the rhesus macaque model of AIDS: the use of simian fibroblasts as an alternative source of target cells for chromium release assays.

The simian immunodeficiency virus (SIV) model of AIDS is widely used for the development of human immunodeficiency virus (HIV) vaccine strategies, particularly for the analysis of correlates of protective immunity. As it is not always possible to establish autologous B-lymphoblastoid cell lines (B-LCL) for use as targets in the analysis of cytotoxic T cell (CTL) activity, we have compared B-LCL with primary simian skin cells. Using a well-defined SIV gag-encoded CTL epitope restricted by Mamu A*01 major histocompatibility complex (MHC) class I, we have shown that peripheral blood mononuclear cells (PBMC) from vaccinated and infected macaques can kill MHC class I-matched skin fibroblasts presenting the cognate epitope but that skin fibroblasts are a less sensitive target than B-LCL for the detection of CTL.