Marjon Navis

Immune escape from HIV-specific antibody-dependent cellular cytotoxicity (ADCC) pressure

Effective immunity to HIV is poorly understood. In particular, a role for antibody-dependent cellular cytotoxicity (ADCC) in controlling HIV is controversial. We hypothesized that significant pressure from HIV-specific ADCC would result in immune-escape variants. A series of ADCC epitopes in HIV-infected subjects to specific consensus strain HIV peptides were mapped using a flow cytometric assay for natural killer cell activation. We then compared the ADCC responses to the same peptide epitope derived from the concurrent HIV sequence(s) expressed in circulating virus. In 9 of 13 epitopes studied, ADCC antibodies were unable to recognize the concurrent HIV sequence. Our studies suggest ADCC responses apply significant immune pressure on the virus. This result has implications for the induction of ADCC responses by HIV vaccines.

Kaposi's sarcoma-associated herpesvirus-encoded G protein-coupled receptor ORF74 constitutively activates p44/p42 MAPK and Akt via G(i) and phospholipase C-dependent signaling pathways

ABSTRACT The G protein-coupled receptor encoded by Kaposis sarcoma-associated herpesvirus, also referred to as ORF74, has been shown to stimulate oncogenic and angiogenic signaling pathways in a constitutively active manner. The biochemical routes linking ORF74 to these signaling pathways are poorly defined. In this study, we show that ORF74 constitutively activates p44/p42 mitogen-activated protein kinase (MAPK) and Akt via Gi- and phospholipase C (PLC)-mediated signaling pathways. Activation of Akt by ORF74 appears to be phosphatidylinositol 3-kinase (PI3-K) dependent but, interestingly, is also mediated by activation of protein kinase C (PKC) and p44/p42 MAPK. ORF74 may signal to Akt via p44/p42 MAPK, which can be activated by Gi, through activation of PI3-K or through PKC via the PLC pathway. Signaling of ORF74 to these proliferative and antiapoptotic signaling pathways can be further modulated positively by growth-related oncogene (GROα/CXCL1) and negatively by human gamma interferon-inducible protein 10 (IP-10/CXCL10), thus acting as an agonist and an inverse agonist, respectively. Despite the ability of the cytomegalovirus-encoded chemokine receptor US28 to constitutively activate PLC, this receptor does not increase phosphorylation of p44/p42 MAPK or Akt in COS-7 cells. Hence, ORF74 appears to signal through a larger diversity of G proteins than US28, allowing it to couple to proliferative and antiapoptotic signaling pathways. ORF74 can therefore be envisioned as an attractive target for novel treatment of Kaposis sarcoma.

Specific antibody‐dependent cellular cytotoxicity responses associated with slow progression of HIV infection

Antibody‐dependent cellular cytotoxicity (ADCC) is potentially an effective adaptive immune response to HIV infection. However, little is understood about the role of ADCC in controlling chronic infection in the small number of long‐term slow‐progressors (LTSP) who maintain a relatively normal immunological state for prolonged periods of time. We analysed HIV‐specific ADCC responses in sera from 139 HIV+ subjects not on antiretroviral therapy. Sixty‐five subjects were LTSP, who maintained a CD4 T‐cell count > 500/μl for over 8 years after infection without antiretroviral therapy and 74 were non‐LTSP individuals. The ADCC responses were measured using an natural killer cell activation assay to overlapping HIV peptides that allowed us to map ADCC epitopes. We found that although the magnitude of ADCC responses in the LTSP cohort were not higher and did not correlate with CD4 T‐cell depletion rates, the LTSP cohort had significantly broader ADCC responses compared with the non‐LTSP cohort. Specifically, regulatory/accessory HIV‐1 proteins were targeted more frequently by LTSP. Indeed, three particular ADCC epitopes within the Vpu protein of HIV were recognized only by LTSP individuals. Our study provides evidence that broader ADCC responses may play a role in long‐term control of HIV progression and suggests novel vaccine targets.

Activation of NK cells by ADCC antibodies and HIV disease progression

Antibody-dependent cellular cytotoxicity (ADCC) is of considerable interest as an immune response that may facilitate the control of HIV infection. We studied ADCC responses prospectively in a cohort of 79 HIV-positive subjects followed up for a mean of 2.3 years without antiretroviral therapy. We used a novel assay of the ability of ADCC to activate natural killer (NK) cells, either from the same HIV-positive subject or from a healthy blood donor. We found that ADCC responses to either gp140 Env protein or HIV peptide pools were common in HIV-positive subjects when NK cells from the HIV-positive subject were used but did not correlate with markers of HIV disease progression. In contrast, ADCC responses to whole gp140 Env protein were strongly associated with a slower decline in CD4 T-cell loss when healthy donor NK cells were used as effectors. Our data had implications for induction of the most effective ADCC responses by HIV vaccines.

HIV Infection Abrogates the Functional Advantage of Natural Killer Cells Educated through KIR3DL1/HLA-Bw4 Interactions To Mediate Anti-HIV Antibody-Dependent Cellular Cytotoxicity

ABSTRACT Combinations of KIR3DL1 and HLA-Bw4 alleles protect against HIV infection and/or disease progression. These combinations enhance NK cell responsiveness through the ontological process of education. However, educated KIR3DL1+ NK cells do not have enhanced degranulation upon direct recognition of autologous HIV-infected cells. Since antibody-dependent cellular cytotoxicity (ADCC) is associated with improved HIV infection outcomes and NK cells overcome inhibition through killer cell immunoglobulin-like receptors (KIR) to mediate ADCC, we hypothesized that KIR3DL1-educated NK cells mediate anti-HIV ADCC against autologous cells. A whole-blood flow cytometry assay was used to evaluate ADCC-induced activation of NK cells. This assay assessed activation (gamma interferon [IFN-γ] production and/or CD107a expression) of KIR3DL1+ and KIR3DL1− NK cells, from HLA-Bw4+ and HLA-Bw4− HIV-positive and HIV-negative individuals, in response to autologous HIV-specific ADCC targets. KIR3DL1+ NK cells were more functional than KIR3DL1− NK cells from HLA-Bw4+, but not HLA-Bw4−, healthy controls. In HIV-infected individuals, no differences in NK cell functionality were observed between KIR3DL1+ and KIR3DL1− NK cells in HLA-Bw4+ individuals, consistent with dysfunction of NK cells in the setting of HIV infection. Reflecting the partial normalization of NK cell responsiveness following initiation of antiretroviral therapy, a significant correlation was observed between the peripheral CD4+ T-lymphocyte counts in antiretroviral therapy-treated subjects and the functionality of NK cells. However, peripheral CD4+ T-lymphocyte counts were not correlated with an anti-HIV ADCC functional advantage in educated KIR3DL1+ NK cells. The abrogation of the functional advantage of educated NK cells may enhance HIV disease progression. Strategies to enhance the potency of NK cell-mediated ADCC may improve HIV therapies and vaccines.

Pol as a target for antibody dependent cellular cytotoxicity responses in HIV-1 infection

Antibody-dependent cellular cytotoxicity (ADCC) may assist in preventing HIV or delaying disease progression. Most prior studies have analysed Env-specific ADCC responses. We hypothesized that effective ADCC-based immunity may target conserved internal viral proteins such as Pol. We analysed the ability overlapping Pol peptides to induce activation of NK cells via ADCC. We prospectively studied ADCC responses in 83 HIV+ subjects followed for 3 years. Pol peptides were commonly targeted by ADCC responses in these chronically infected subjects (in 32 of the 83 subjects). However, Pol-specific ADCC responses declined over time and did not correlate with delayed HIV progression, measured by either baseline CD4 T cells, CD4 T cell loss over time, baseline viral load or the need to start antiretroviral therapy. Although Pol is frequently targeted by ADCC in HIV+ subjects, the strength or specificity of Pol-specific ADCC responses needs to be modulated to be effective in delaying HIV progression.

Activation of NK Cells by ADCC Responses During Early HIV Infection

Partial control of HIV occurs during acute infection, although the mechanisms responsible are poorly understood. We studied the ability of antibody-dependent cellular cytotoxicity (ADCC) antibodies in serum to activate natural killer (NK) cells in longitudinal samples from 8 subjects with well-defined early HIV infection who controlled viremia to low levels. NK cell activation by ADCC antibodies to gp140 Env proteins was detected in half of the subjects at the first time point studied, a mean of 111 d after the estimated time of infection. In contrast, ADCC-mediated NK cell activation in response to linear HIV peptides evolved more slowly, over the first 2 y of infection. Our studies suggest that HIV-specific ADCC responses to conformational epitopes occur early during acute HIV infection, and broaden to include linear epitopes over time. These findings have implications for the immune control of HIV.

Downregulation of Interleukin-18-Mediated Cell Signaling and Interferon Gamma Expression by the Hepatitis B Virus e Antigen

ABSTRACT The mechanisms by which hepatitis B virus (HBV) establishes and maintains chronic hepatitis B infection (CHB) are poorly defined. Innate immune responses play an important role in reducing HBV replication and pathogenesis. HBV has developed numerous mechanisms to escape these responses, including the production of the secreted hepatitis B e antigen (HBeAg), which has been shown to regulate antiviral toll-like receptor (TLR) and interleukin-1 (IL-1) signaling. IL-18 is a related cytokine that inhibits HBV replication in hepatoma cell lines and in the liver through the induction of gamma interferon (IFN-γ) by NK cells and T cells. We hypothesized that HBV or HBV proteins inhibit IFN-γ expression by NK cells as an accessory immunomodulatory function. We show that HBeAg protein inhibits the NF-κB pathway and thereby downregulates NK cell IFN-γ expression. Additionally, IFN-γ expression was significantly inhibited by exposure to serum from individuals with HBeAg-positive but not HBeAg-negative chronic HBV infection. Further, we show that the HBeAg protein suppresses IL-18-mediated NF-κB signaling in NK and hepatoma cells via modulation of the NF-κB pathway. Together, these findings show that the HBeAg inhibits IL-18 signaling and IFN-γ expression, which may play an important role in the establishment and/or maintenance of persistent HBV infection. IMPORTANCE It is becoming increasingly apparent that NK cells play a role in the establishment and/or maintenance of chronic hepatitis B infection. The secreted HBeAg is an important regulator of innate and adaptive immune responses. We now show that the HBeAg downregulates NK cell-mediated IFN-γ production and IL-18 signaling, which may contribute to the establishment of infection and/or viral persistence. Our findings build on previous studies showing that the HBeAg also suppresses the TLR and IL-1 signaling pathways, suggesting that this viral protein is a key regulator of antiviral innate immune responses.

Anti-HIV-1 antibody-dependent cellular cytotoxicity mediated by hyperimmune bovine colostrum IgG

Antibodies with antibody‐dependent cellular cytotoxicity (ADCC) activity play an important role in protection against HIV‐1 infection, but generating sufficient amounts of antibodies to study their protective efficacy is difficult. HIV‐specific IgG can be easily and inexpensively produced in large quantities using bovine colostrum. We previously vaccinated cows with HIV‐1 envelope gp140 and elicited high titers of anti‐gp140‐binding IgG in colostrum. In the present study, we determined whether bovine antibodies would also demonstrate specific cytotoxic activity. We found that bovine IgG bind to Fcγ‐receptors (FcγRs) on human neutrophils, monocytes, and NK cells in a dose‐dependent manner. Antibody‐dependent killing was observed in the presence of anti‐HIV‐1 colostrum IgG but not nonimmune colostrum IgG. Killing was dependent on Fc and FcγR interaction since ADDC activity was not seen with F(ab’)2 fragments. ADCC activity was primarily mediated by CD14+ monocytes with FcγRIIa (CD32a) as the major receptor responsible for monocyte‐mediated ADCC in response to bovine IgG. In conclusion, we demonstrate that bovine anti‐HIV colostrum IgG have robust HIV‐1‐specific ADCC activity and therefore offer a useful source of antibodies able to provide a rapid and potent response against HIV‐1 infection. This could assist the development of novel Ab‐mediated approaches for prevention of HIV‐1 transmission.

Activation of NK cells by HIV-specific ADCC antibodies: role for granulocytes in expressing HIV-1 peptide epitopes.

HIV-specific ADCC antibodies could play a role in providing protective immunity. We have developed a whole blood ADCC assay that measures NK cell activation in response to HIV peptide epitopes. These HIV peptide-specific ADCC responses are associated with escape from immune recognition and slower progression of HIV infection and represent interesting HIV vaccine antigens. However, the mechanism by which these epitopes are expressed and whether or not they induce NK-mediated killing of cells expressing such peptide-antigens is not understood. Herein, we show that fluorescent-tagged ADCC peptide epitopes associate with blood granulocytes. The peptide-associated granulocytes become a specific target for antibody-mediated killing, as shown by enhanced expression of apoptosis marker Annexin and reduction in cell numbers. When HIV Envelope gp140 protein is utilized in the ADCC assay, we detected binding to its ligand, CD4. During the incubation, cells co-expressing gp140 and CD4 reduce in number. We also detected increasing Annexin expression in these cells. These data indicate that blood cells expressing HIV-specific ADCC epitopes are targeted for killing by NK cells in the presence of ADCC antibodies in HIV+ plasma and provide a clearer framework to evaluate these antigens as vaccine candidates.