A novel mechanism linking memory stem cells with innate immunity in protection against HIV-1 infection.

HIV infection affects 37 million people and about 1.7 million are infected annually. Among the phase III clinical trials only the RV144 vaccine trial elicited significant protection against HIV-1 acquisition, but the efficacy and immune memory were inadequate. To boost these vaccine functions we studied T stem cell memory (TSCM) and innate immunity. TSCM cells were identified by phenotypic markers of CD4+ T cells and they were further characterised into 4 subsets. These expressed the common IL-2/IL-15 receptors and another subset of APOBEC3G anti-viral restriction factors, both of which were upregulated. In contrast, CD4+ TSCM cells expressing CCR5 co-receptors and α4ß7 mucosal homing integrins were decreased. A parallel increase in CD4+ T cells was recorded with IL-15 receptors, APOBEC3G and CC chemokines, the latter downmodulating CCR5 molecules. We suggest a novel mechanism of dual memory stem cells; the established sequential memory pathway, TSCM →Central →Effector memory CD4+ T cells and the innate pathway consisting of the 4 subsets of TSCM. Both pathways are likely to be activated by endogenous HSP70. The TSCM memory stem cell and innate immunity pathways have to be optimised to boost the efficacy and immune memory of protection against HIV-1 in the clinical trial.

Potential molecular mimicry between the human endogenous retrovirus W family envelope proteins and myelin proteins in multiple sclerosis.

Multiple sclerosis is an autoimmune disease caused by the destruction of the myelin sheath in the central nervous system. The major target molecules for the immune response are the myelin basic protein, myelin oligodendrocyte glycoprotein and proteolipid protein but the aetiology of the disease is as yet poorly understood. The HLA Class II allele DRB1*1501 in particular as well as DRB5*0101 and the expression of human endogenous retroviral envelope proteins have been linked to multiple sclerosis but the molecular mechanisms relating these remain to be elucidated. We hypothesised that cross-reactive peptide epitopes in retroviral envelope proteins and myelin proteins that can be presented by the two Class II DR molecules may play a role in initiating multiple sclerosis. Sequence homologies between retroviral envelope and myelin proteins and in silico predictions of peptides derived from them that are able to bind to the two Class II alleles were examined to test the hypothesis. The results support the hypothesis that molecular mimicry in peptide epitopes from envelope proteins of the HERV-W family of endogenous retroviruses and myelin proteins is possible and could potentially trigger multiple sclerosis. Mimicry between syncytin-1, a HERV-W envelope protein that is expressed during placentation, and myelin proteins may also explain the higher prevalence of multiple sclerosis in women. Experiments to test the ability of the identified peptide epitopes to activate TH cells are required to confirm the present findings.

The role of innate APOBEC3G and adaptive AID immune responses in HLA-HIV/SIV immunized SHIV infected macaques.

The AID/APOBEC family (activation induced deaminase/apolipoprotein B mRNA editing cytokine deaminase) in B cells play important roles in adaptive and innate immunity. Whereas APOBEC3G has been studied in CD4+ T cells and myeloid cells its functional potential in B cells has received little attention. AID combines two critical functions of antibodies, class switching and affinity maturation and may serve as a functional surrogate of protection. These functions were studied following systemic immunization of rhesus macaques with recombinant HLA constructs, linked with HIV and SIV antigens and HSP70 to dextran. The results showed significant upregulation of AID in CD20+ B cells, APOBEC 3G in CD27+ memory B cells and CD4+ effector memory T cells. After immunization the upregulated APOBEC 3G and AID were directly correlated in B cells (p<0.0001). Following challenge with SHIV SF162.P4 the viral load was inversely correlated with AID in B cells and APOBEC 3G in B and T cells, suggesting that both deaminases may have protective functions. Investigation of major interactions between DC, T cells and B cells showed significant increase in membrane associated IL-15 in DC and CD40L in CD4+ T cells. IL-15 binds the IL-15 receptor complex in CD4+ T and B cells, which may reactivate the DC, T and B cell interactions. The overall results are consistent with AID inhibiting pre-entry SHIV by eliciting IgG and IgA antibodies, whereas APOBEC 3G may contribute to the post-entry control of SHIV replication and cellular spread.

B-cell agonists up-regulate AID and APOBEC3G deaminases, which induce IgA and IgG class antibodies and anti-viral function.

B cells express two critical deaminases in the development of adaptive and innate immunity. Activation-induced cytidine deaminase (AID) functions in class switch recombination, somatic hypermutation and may result in affinity maturation of antibodies. Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G; A3G) is an innate anti-retroviral factor that inhibits HIV replication. We have studied a number of B-cell agonists with the aim of identifying the most effective agents that will up-regulate both deaminases and thereby enhance adaptive and innate immunity. CD40 ligand (CD40L) with interleukin-4 or HLA-class II antibodies significantly up-regulated both AID and A3G in isolated human CD19(+) B cells. The functions of these deaminases were demonstrated by enhancement of B-cell surface expression of IgA and IgG and inducing significantly higher IgA and IgG4 antibodies. An enhanced A3G function was then demonstrated by inhibition of HIV-1 replication in co-culture of CD4(+) T cells with autologous B cells, treated with CD40L and CD4 or HLA antibodies, compared with unstimulated human B cells. The dual B-cell-induced deaminase functions may be critical in IgA and IgG antibodies inhibiting pre-entry and A3G that of post-entry HIV-1 transmission and suggests a novel strategy of immunization, especially relevant to mucosal infections.

A recombinant human HLA-class I antigen linked to dextran elicits innate and adaptive immune responses.

The objective of this study was to produce and evaluate the immunogenic potential of a recombinant HLA-class I antigen linked to dextran. The HLA-A*0201 heavy chain and beta2 microglobulin were cloned by PCR amplification of overlapping oligonucleotides and produced in E. coli. These were assembled with a CMV binding peptide motif, the HLA complex was biotinylated and bound by streptavidin coated dextran at a ratio of 24 HLA to 1 dextran molecule (termed Dextramer). Allostimulation of human PBMC in vitro and in vivo immunization of Balb c mice with the HLA-A*0201 construct elicited CD4+ and CD8+ T cell proliferative responses, IgG specific antibodies in mice and in human T cell proliferation and APOBEC3G mRNA. These adaptive and innate immune responses induced by a novel recombinant HLA construct in human cells and mice suggest their application as a potential vaccine candidate against HIV infection.

Stress-activated dendritic cells interact with CD4+ T cells to elicit homeostatic memory.

Evidence is presented that thermal or oxidizing stress-activated DC interact with CD4(+) T cells to induce and maintain a TCR-independent homeostatic memory circuit. Stress-activated DC expressed endogenous intra-cellular and cell surface HSP70. The NF-kappaB signalling pathway was activated and led to the expression of membrane-associated IL-15 molecules. These interacted with the IL-15 receptor complex on CD4(+) T cells, thus activating the Jak3 and STAT5 phosphorylation signalling pathway to induce CD40 ligand expression, T-cell proliferation and IFN-gamma production. CD40 ligand on CD4(+) T cells in turn re-activated CD40 molecules on DC, inducing DC maturation and IL-15 expression thereby maintaining the feedback circuit. The proliferating CD4(+) T cells were characterized as CD45RA(-) CD62L(+) central memory cells, which underwent homeostatic proliferation. The circuit is independent of antigen and MHC-class-II-TCR interaction as demonstrated by resistance to TCR inhibition by ZAP70 inhibitor or MHC-class II antibodies. These findings suggest that stress can activate a DC-CD4(+) T-cell interacting circuit, which may be responsible for maintaining a homeostatic antigen-independent memory.

Mucosal immunization in macaques upregulates the innate APOBEC 3G anti-viral factor in CD4(+) memory T cells.

APOBEC3G is an innate intracellular anti-viral factor which deaminates retroviral cytidine to uridine. In vivo studies of APOBEC3G (A3G) were carried out in rhesus macaques, following mucosal immunization with SIV antigens and CCR5 peptides, linked to the 70kDa heat shock protein. A progressive increase in A3G mRNA was elicited in PBMC after each immunization (p<0.0002 to p< or =0.02), which was maintained for at least 17 weeks. Analysis of memory T cells showed a significant increase in A3G mRNA and protein in CD4(+)CCR5(+) memory T cells in circulating (p=0.0001), splenic (p=0.0001), iliac lymph nodes (p=0.002) and rectal (p=0.01) cells of the immunized compared with unimmunized macaques. Mucosal challenge with SIVmac 251 showed a significant increase in A3G mRNA in the CD4(+)CCR5(+) circulating cells (p<0.01) and the draining iliac lymph node cells (p<0.05) in the immunized uninfected macaques, consistent with a protective effect exerted by A3G. The results suggest that mucosal immunization in a non-human primate can induce features of a memory response to an innate anti-viral factor in CCR5(+)CD4(+) memory and CD4(+)CD95(+)CCR7(-) effector memory T cells.

Stimulation of cell surface CCR5 and CD40 molecules by their ligands or by HSP70 up-regulates APOBEC3G expression in CD4(+) T cells and dendritic cells.

Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like-3G (A3G) is an intracellular innate antiviral factor that deaminates retroviral cytidine to uridine. In an attempt to harness the anti-HIV effect of A3G, we searched for an agent that would up-regulate A3G and identify the receptors involved. Stimulation of cell surface CCR5 with CCL3 and CD40 with CD40L or both molecules with microbial 70-kDa heat shock protein (HSP)70 up-regulated A3G mRNA and protein expression in human CD4(+) T cells and monocyte-derived dendritic cells (DC), demonstrated by real-time PCR and Western blots, respectively. The specificity of CCR5 and CD40 stimulation was established by inhibition with TAK 779 and mAb to CD40, as well as using human embryonic kidney 293 cells transfected with CCR5 and CD40, respectively. A dose-dependent increase of A3G in CCL3- or HSP70-stimulated CD4(+) T cells was associated with inhibition in HIV-1 infectivity. To differentiate between the inhibitory effect of HSP70-induced CCR5 binding and that of A3G, GFP-labeled pseudovirions were used to infect human embryonic kidney 293 cells, which showed inhibition of pseudovirion uptake, consistent with A3G being responsible for the inhibitory effect. Ligation of cell surface CCR5 receptors by CCL3 or CD40 by CD40L activated the ERK1/2 and p38 MAPK signaling pathways that induced A3G mRNA expression and production of the A3G protein. These in vitro results were corroborated by in vivo studies in rhesus macaques in which A3G was significantly up-regulated following immunization with SIVgp120 and p27 linked to HSP70. This novel preventive approach may in addition to adaptive immunity use the intracellular innate antiviral effect of A3G.

Interaction between the CCR5 chemokine receptors and microbial HSP70.

Evidence is presented that the microbial 70-kD heat shock protein (HSP70) binds to CCR5 chemokine receptors in CCR5-transfected cell lines and in primary human cells. Significant CCR5-mediated calcium mobilization was stimulated by HSP70 and inhibited with TAK 779, which is a specific CCR5 antagonist. HSP70-mediated activation of the p38 MAPK phosphorylation signaling pathway was also demonstrated in CCR5-transfected HEK 293 cells. Direct binding of three extracellular peptides of CCR5 to HSP70 was demonstrated by surface plasmon resonance. Functional evidence of an interaction between HSP70, CCR5 and CD40 was shown by enhanced production of CCL5 by HEK 293 cells transfected with both CD40 and CCR5. Primary monocyte-derived immature DC stimulated with HSP70 produced IL-12 p40, which showed dose-dependent inhibition of >90% on treatment with both TAK 779 and anti-CD40 mAb. Stimulation of IL-12 p40 or TNF-alpha by HSP70 was related to the differential cell surface expression of CCR5 in primary human immature and mature DC, and those with the homozygous triangle DeltaDelta32 CCR5 mutation. These findings may be of significance in the interaction between HSP70 and immune responses of CCR5+ T cells in HIV-1 infection, as well as in inflammatory bowel disease.

Identification of stimulating and inhibitory epitopes within the heat shock protein 70 molecule that modulate cytokine production and maturation of dendritic cells.

The 70-kDa microbial heat shock protein (mHSP70) has a profound effect on the immune system, interacting with the CD40 receptor on DC and monocytes to produce cytokines and chemokines. The mHSP70 also induces maturation of dendritic cells (DC) and thus acts as an alternative ligand to CD40L on T cells. In this investigation, we have identified a cytokine-stimulating epitope (peptide 407-426), by activating DC with overlapping synthetic peptides (20-mers) derived from the sequence of mHSP70. This peptide also significantly enhances maturation of DC stimulated by mHSP70 or CD40L. The epitope is located at the base of the peptide-binding groove of HSP70 and has five critical residues. Furthermore, an inhibitory epitope (p457-496) was identified downstream from the peptide-binding groove that inhibits cytokine production and maturation of DC stimulated by HSP70 or CD40L. The p38 MAP kinase phosphorylation is critical in the alternative CD40-HSP70 pathway and is inhibited by p457-496 but enhanced by p407-426.

Effect of heterosexual intercourse on mucosal alloimmunisation and resistance to HIV-1 infection.

BACKGROUND: Unprotected sexual intercourse between regular heterosexual partners could elicit alloimmune responses that might be associated with inhibition of in-vitro HIV-1 infectivity. We investigated this hypothesis in people practising unprotected sex and those using protection. METHODS: We recruited 82 participants from an outpatient genitourinary medicine clinic. 29 monogamous heterosexual couples having unprotected sex; and 15 women and 10 men having condom protected or no sex. We used the mixed leucocyte reaction (MLR), stimulating one partner's peripheral blood mononuclear cells (PBMC) with the other partner's irradiated PBMC and compared the resulting response with control PBMC. We studied resistance to HIV-1 infection by challenging activated CD4-positive T cells with CCR5-binding and CXCR4-binding HIV-1 strains, and comparing the infectivity in participants having unprotected sex with those practising protected sex. We used the correlation coefficient to establish the significance of the relation between MLR and HIV-1 infectivity. FINDINGS: We recorded a significant increase in the stimulation indices in PBMC from women whose cells were stimulated with irradiated PBMC (2%, 10%, or 50%) from their regular partners. The mean with 10% partner's cells was 8.6 (SD 7.7), compared with those from unrelated cells (4.7 [3.9], p=0.009). Significant alloimmune responses were also seen in corresponding male partners, but only with 50% stimulating cells (p=0.013). Dose-dependent inhibition of activated CD4-positive T cells to HIV-1 infection with both binding strains was noted in vitro in women practising unprotected intercourse, compared with those having protected sex or having no sex for more than 1 year. Highly significant differences were found for CCR5 (p=0.0001) and for CXCR4 (p=0.001) strains of HIV-1 at all four virus-concentrations. Male partners also showed in-vitro inhibition of HIV-1 but this was less than that in women. INTERPRETATION: Unprotected sexual intercourse might result in alloimmunisation stimulated by HLA antigens in seminal or cervicovaginal fluid. Mucosal alloimmunisation may reduce infection by HIV-1, and the role of such immunisation in preventive and therapeutic vaccination should be investigated.

A comparative investigation of CC chemokines and SIV suppressor factors generated by CD8+ and CD4+ T cells and CD14+ monocytes.

The capacity of CD8+ and CD4+ T cells and CD14+ monocytes to generate the CC chemokines, RANTES, MIP-1alpha and MIP-1beta, and SIV suppressor factors were studied using cells separated from PBMC of macaques immunized with the 70-kDa heat shock protein (HSP70). Unimmunized macaques showed low levels of the three CC chemokines and SIV-SF, and they showed little variation between PBMC and the two subsets of T cells stimulated with PHA. Immunization with HSP70 elicited an increase in the in vitro concentration of each of the three CC chemokines and SF. This was found with PBMC, CD4+ and CD8+ T cells and to a lesser extent with monocytes, when conventionally separated enriched cell subsets were examined from the same PBMC. However, the concentrations of the three CC chemokines derived from highly purified cell-sorted populations (>95%) were greatly increased, as compared with the enriched cell subsets. The concentration of each of the three chemokines was highest for CD8+ T cells, decreased with CD4+ T cells and was lowest with the CD14+ monocytes, but the latter were not stimulated. Neutralization assays with antibodies to the three CC chemokines showed that the antiviral activity generated by the four populations of cells could be largely accounted for by the three CC chemokines. The results of this comparative study suggests that CD8+ as well as CD4+ T cells and CD14+ monocytes generate the three CC chemokines and SIV-SF when stimulated with a mitogen, and that the baseline innate level can be upregulated by adaptive immune responses to a specific antigen.