Kaboutar Babaahmady

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 partners peripheral blood mononuclear cells (PBMC) with the other partners 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% partners 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.

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.

The emerging role of innate immunity in protection against HIV-1 infection.

Preventive immunization against HIV-1 infection requires a rapid immune response that does not rely exclusively on B or T cell memory. Innate immunity may fulfill this function as it may be activated directly at the time of HIV-1 transmission, inhibit early HIV-1 replication, stimulate adaptive immunity and enable specific antibodies followed by CD8(+) T cells to deal with the virus effectively. The three components of innate immunity - cellular, extracellular and intracellular - are presented, with an example given for each of these components; gammadelta T cells, CC chemokines and APOBEC3G. This brief account is presented to highlight the immuno-virological concept of coordinating activated innate immunity with adaptive antibody and T cell responses in preventive vaccination against HIV-1 infection.

Immunogenicity of the Extracellular Domains of C-C Chemokine Receptor 5 and the In Vitro Effects on Simian Immunodeficiency Virus or HIV Infectivity

The C-C chemokine receptor CCR5 serves an important function in chemotaxis of lymphocytes, monocytes, and dendritic cells. CCR5 is also the major coreceptor in most macrophage-tropic HIV-1 infections. Immunization of rhesus macaques with a baculovirus-generated CCR5 construct or peptides derived from the sequences of the four extracellular domains of CCR5 elicited IgG and IgA Abs, inhibition of SIV replication, and CD4+ T cell proliferative responses to three of the extracellular domains of CCR5. The immune sera reacted with cell surface CCR5 expressed on HEK 293 cells. T and B cell epitope mapping revealed major and minor T and B cell epitopes in the N-terminal, first, and second loops of CCR5. The three C-C chemokines, RANTES, macrophage-inflammatory protein-1α, and macrophage-inflammatory protein-1β, were up-regulated by immunization with the CCR5-derived peptides, and the cell surface expression of CCR5 was decreased. The CCR5 Abs were complementary to the C-C chemokines in inhibiting HIV replication in vitro. Immunization with the four extracellular domains of CCR5 suggests that three of them are immunogenic, with maximal T cell responses being elicited by the second loop peptide. However, maximal Abs to the cell surface CCR5 or viral inhibitory Abs in vitro were induced by the N-terminal peptide. Up-regulation of the three C-C chemokines and down-modulation of cell surface CCR5 were elicited by the second loop, N-terminal, and first loop peptides. The data suggest that a dual mechanism of C-C chemokines and specific Abs may engage and down-modulate the CCR5 coreceptors and prevent in vitro HIV or SIV replication.

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‐κB 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‐γ 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.

Inhibition of Human Immunodeficiency Virus Type 1 Infection of Human CD4+ T Cells by Microbial HSP70 and the Peptide Epitope 407-426

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) virions contain heat shock proteins (HSP), but these proteins have received limited attention. The objectives of this study were to establish if the microbial 70-kDa HSP exerts an inhibitory effect on the HIV-1 infection of human CD4+ T cells, to identify an inhibitory peptide epitope within the sequence of HSP70, and to evaluate the kinetic features of any inhibitory activity. The results of these studies suggest that microbial HSP70 exerts dose-dependent inhibition on CCR5 (R5) strains of clades B, C, and D of HIV-1 infecting human CD4+ T cells. The site of the HIV-1-inhibitory function was identified within the C-terminal peptide binding domain of HSP70, and the function is expressed by the peptide epitope comprising amino acids 407 to 426. The mechanism of inhibition of HIV-1 infectivity by HSP70 is blocking of the CCR5 coreceptors directly and indirectly by inducing CC chemokines and APOBEC3G. The inhibitory effect of HSP70, its C-terminal fragment, or peptide 407-426 may make HSP70 useful as a microbicidal agent. A potentiating noncognate inhibition of HIV-1 infectivity by combined treatment with HSP70 and monoclonal or polyclonal antibody to CCR5 was demonstrated. This novel strategy may be utilized in therapeutic immunization against HIV-1 infection.

Immunization with recombinant macaque major histocompatibility complex class I and II and human immunodeficiency virus gp140 inhibits simian-human immunodeficiency virus infection in macaques.

Genetic, epidemiological and experimental evidence suggest that the major histocompatibility complex (MHC) is critical in controlling human immunodeficiency virus (HIV) infection. The objectives of this study were to determine whether novel recombinant Mamu MHC constructs would elicit protection against rectal challenge with heterologous simian-human immunodeficiency virus (SHIV) strain SF162.P4 in rhesus macaques. Mamu class I and II gene products were linked together with HIV gp140, simian immunodeficiency virus (SIV) p27 and heat-shock protein 70 to dextran. The vaccine was administered to two groups, each consisting of nine macaques, either subcutaneously (SC), or rectally and boosted by SC immunization. The controls were untreated or adjuvant-treated animals. Repetitive rectal challenges with up to ten doses of SHIV SF162.P4 showed a significant decrease in the peak and sequential viral RNA concentrations, and three macaques remained uninfected, in the nine SC-immunized animals, compared with infection in all nine controls. Macaques immunized rectally followed by SC boosters showed a less significant decrease in both sequential and peak viral loads compared with the SC-immunized animals, and all were infected following rectal challenge with SHIV SF162.P4. Plasma and mucosal IgG and IgA antibodies to Mamu class I alleles and HIV gp120, as well as to RANTES (regulated upon activation, normal T-cell expressed, and secreted; CCR5) were increased, and showed significant inverse correlations with the peak viral load. These results suggested that allo-immunization with recombinant MHC constructs linked to HIV-SIV antigens merits further investigation in preventing HIV-1 infection.

Effect of Vaginal Immunization with HIVgp140 and HSP70 on HIV-1 Replication and Innate and T Cell Adaptive Immunity in Women

ABSTRACT The international effort to prevent HIV-1 infection by vaccination has failed to develop an effective vaccine. The aim of this vaccine trial in women was to administer by the vaginal mucosal route a vaccine consisting of HIV-1 gp140 linked to the chaperone 70-kDa heat shock protein (HSP70). The primary objective was to determine the safety of the vaccine. The secondary objective was to examine HIV-1 infectivity ex vivo and innate and adaptive immunity to HIV-1. Protocol-defined female volunteers were recruited. HIV-1 CN54gp140 linked to HSP70 was administered by the vaginal route. Significant adverse reactions were not detected. HIV-1 was significantly inhibited ex vivo in postimmunization CD4+ T cells compared with preimmunization CD4+ T cells. The innate antiviral restrictive factor APOBEC3G was significantly upregulated, as were CC chemokines which induce downregulation of CCR5 in CD4+ T cells. Indeed, a significant inverse correlation between the proportion of CCR5+ T cells and the concentration of CCL-3 or CCL-5 was found. Importantly, the upregulation of APOBEC3G showed a significant inverse correlation, whereas CCR5 exhibited a trend to correlate with inhibition of HIV-1 infection (r = 0.51). Furthermore, specific CD4+ and CD8+ T cell proliferative responses were significantly increased and CD4+ T cells showed a trend to have an inverse correlation with the viral load (r = −0.60). However, HIVgp140-specific IgG or IgA antibodies were not detected. The results provide proof of concept that an innate mechanism consisting of CC chemokines, APOBEC3G, and adaptive immunity by CD4 and CD8 T cells might be involved in controlling HIV-1 infectivity following vaginal mucosal immunization in women. (This study has been registered at ClinicalTrials.gov under registration no. NCT01285141.) IMPORTANCE Vaginal immunization of women with a vaccine consisting of HIVgp140 linked to the 70-kDa heat shock protein (HSP70) elicited ex vivo significant inhibition of HIV-1 replication in postimmunization CD4+ T cells compared with that in preimmunization peripheral blood mononuclear cells. There were no significant adverse events. The vaccine induced the significant upregulation of CC chemokines and the downmodulation of CCR5 expression in CD4+ T cells, as well as an inverse correlation between them. Furthermore, the level of CCR5 expression was directly correlated with the viral load, consistent with the protective mechanism in which a decrease in CCR5 molecules on CD4+ T cells decreases HIV-1 envelope binding. Expression of the antiviral restriction factor APOBEC3G was inversely correlated with the viral load, suggesting that it may inhibit intracellular HIV-1 replication. Both CD4+ and CD8+ T cells showed HIVgp140- and HSP70-specific proliferation. A strong inverse correlation between the proportion of CC chemokine-modulated CCR5-expressing CD4+ T cells and the stimulation of CD4+ or CD8+ T cell proliferation by HIVgp140 was found, demonstrating a significant interaction between innate and adaptive immunity. This is the first clinical trial of vaginal immunization in women using only HIVgp140 and HSP70 administered by the mucosal route (3 times) in which a dual innate protective mechanism was induced and enhanced by significant adaptive CD4+ and CD8+ T cell proliferative responses.

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.

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.