Th2 polarization in peripheral blood mononuclear cells from human immunodeficiency virus (HIV)-infected subjects, as activated by HIV virus-like particles.

We have recently shown that human immunodeficiency virus type 1 (HIV-1) Pr55(gag) virus-like particles (HIV-VLPs), produced in a baculovirus expression system and presenting a gp120 molecule from a Ugandan HIV-1 isolate of clade A, induce maturation and activation of monocyte-derived dendritic cells (MDDCs) with a production of Th1- and Th2-specific cytokines. Furthermore, HIV-VLP-loaded MDDCs are able to induce a primary and secondary response in autologous human CD4(+) T cells in an ex vivo immunization assay. In the present study, we show that similar data can be obtained directly with fresh peripheral blood mononuclear cells (PBMCs), and the HIV-1 seropositivity status, with either low or high viremia, does not significantly impair the immune activation status and the responsiveness of circulating monocyte CD14(+) cell populations to an immunogenic stimulus. Some HIV-1-seropositive subjects, however, show a complete lack of maturation induced by HIV-VLPs in CD14(+) circulating cells, which does not consistently correlate with an advanced status of HIV-1 infection. The established Th2 polarization in both HIV-seropositive groups is efficiently boosted by HIV-VLP induction and does not switch into a Th1 pattern, strongly suggesting that specific Th1 adjuvants would be required for therapeutic effectiveness in HIV-1-infected subjects. These results indicate the possibility of screening PBMCs for donor susceptibility to an immunogen treatment, which would greatly simplify the identification of "responsive" vaccinees as well as the understanding of eventual failures in individuals enrolled in clinical trials.

Baculovirus-derived human immunodeficiency virus type 1 virus-like particles activate dendritic cells and induce ex vivo T-cell responses.

We have recently developed a candidate human immunodeficiency virus type 1 (HIV-1) vaccine model based on HIV-1 Pr55(gag) virus-like particles (HIV-VLPs), produced in a baculovirus expression system and presenting a gp120 molecule from a Ugandan HIV-1 isolate of clade A (HIV-VLP(A)s). The HIV-VLP(A)s show the induction in BALB/c mice of systemic and mucosal neutralizing antibodies as well as cytotoxic T lymphocytes, by intraperitoneal as well as intranasal administration. In the present article, the effects of the baculovirus-expressed HIV-VLPs on human immature monocyte-derived dendritic cells (MDDCs) have been evaluated. The HIV-VLPs efficiently induce maturation and activation of MDDCs and are incorporated into MDDCs preferentially via an actin-dependent macropinocytosis and endocytosis. The HIV-VLP-activated MDDCs show enhanced Th1- and Th2-specific cytokine production, and the effects of HIV-VLPs on MDDCs are not mediated through Toll-like receptors 2 and 4 (TLR2 and -4) signaling. Finally, HIV-VLP-loaded MDDCs are able to induce a primary and secondary response in autologous human CD4(+) T cells in an ex vivo immunization assay. Our results on the interaction and processing of baculovirus HIV-VLPs by MDDCs give an insight into the mechanisms underlying the immune response induced by HIV-VLP(A)s in vivo.

Rapamycin causes down-regulation of CCR5 and accumulation of anti-HIV beta-chemokines: an approach to suppress R5 strains of HIV-1.

Propagation of R5 strains of HIV-1 on CD4 lymphocytes and macrophages requires expression of the CCR5 coreceptor on the cell surface. Individuals lacking CCR5 (CCR5 Delta 32 homozygous genotype) are phenotypically normal and resistant to infection with HIV-1. CCR5 expression on lymphocytes depends on signaling through the IL-2 receptor. By FACS analysis we demonstrate that rapamycin (RAPA), a drug that disrupts IL-2 receptor signaling, reduces CCR5 surface expression on T cells at concentrations as low as 1 nM. In addition, lower concentrations of RAPA (0.01 nM) were sufficient to reduce CCR5 surface expression on maturing monocytes. PCR analysis on peripheral blood mononuclear cells (PBMCs) showed that RAPA interfered with CCR5 expression at the transcriptional level. Reduced expression of CCR5 on PBMCs cultured in the presence of RAPA was associated with increased extracellular levels of macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta. In infectivity assays, RAPA suppressed the replication of R5 strains of HIV-1 both in PBMC and macrophage cultures. In total PBMC cultures, RAPA-mediated inhibition of CCR5-using strains of HIV-1 occurred at 0.01 nM, a concentration of drug that is approximately 103 times lower than therapeutic through levels of drug in renal transplant recipients. In addition, RAPA enhanced the antiviral activity of the CCR5 antagonist TAK-779. These results suggest that low concentrations of RAPA may have a role in both the treatment and prevention of HIV-1 infection.

Therapeutic vaccine to control stromal tumor-induced immunosuppression in human uterine cervixc cancer.

Cancer cells may escape immune surveillance by secreting in their microenvironment soluble factors that may locally paralyze the stromal effector immune cells. In the human uterine cervix cancer, HPV-16 E7 protein, released in the stroma, should contribute to cancer cells immune escape since this protein inhibits the cellular immune response to recall antigens or alloantigens and strongly enhances the release of immunosuppressive cytokines by APCs. This prompted us to prepare a therapeutic vaccine triggering anti-E7 neutralizing Abs to antagonize the E7-induced stromal immunosuppressive effects and allow cellular immune reaction towards cancer cells including specific CTLs, induced by conventional vaccine, to be effective. Since HPV-16 is a mucosotropic virus, this therapeutic vaccine has been prepared to generate systemic as well as mucosal immunity.

Active versus passive anti-cytokine antibody therapy against cytokine-associated chronic diseases.

Current therapeutic vaccine trials in major chronic diseases including AIDS, cancer, allergy and autoimmunity, target antigenic pathogens but not the pathogenic stromal cytokines which can be major sources of histopathologic processes. Considering that the limited efficacy of these vaccines has been ascribed to local pathogen-induced cytokine dysfunction, we propose to antagonize pathogenic cytokine(s) by high affinity neutralizing auto-Abs triggered by specific anti-cytokine vaccines. As anticipated by theoretical considerations, animal experiments and initial clinical trials showed that anti-cytokine immunization was safe, well tolerated and triggered transient high titers Abs neutralizing pathogenic cytokines but, in contrast to conventional vaccines, no relevant cellular response was observed. Advantages of active versus passive anti-cytokine Ab therapy, particularly for long-term treatments, as those required in AIDS, cancer, allergy and autoimmunity include greater ease of maintaining high Ab titers, lack of anti-antibody responses and low cost.

Targeting Tat and IFN(alpha) as a therapeutic AIDS vaccine.

Evolution to AIDS is characterized by a progressive cellular immune suppression. Although there is substantial evidence for several mechanisms involved in disrupting the immune response by induction of apoptosis in responder cells by contact with infected cells, we propose that humoral factors also play a role, and that one such factor is the extracellular form of the human immunodeficiency virus (HIV)-1 Tat protein and another is IFN(alpha). Both Tat and interferon-alpha (IFN(alpha)) inhibit antigen-stimulate T-cell proliferation, and specific anti-Tat and/or anti-IFN(alpha) Abs prevent generation of HIV-1-induced suppressor cells. We propose that high titer anti-Tat and/or anti-IFN(alpha) Abs, neutralizing extracellular Tat, and/or IFN(alpha), induced by vaccines described here, antagonize HIV-1-induced immunosuppression. Innocuous vaccines were prepared by using inactivated but immunogenic Tat (Toxoid) and inactivated and immunogenic IFN(alpha) (kinoid) derivatives. Both Tat Toxoid and IFN(alpha) kinoid were well tolerated and elicited specific neutralizing antibodies (Abs) in mice, monkeys, and seronegative and HIV-1-infected individuals.

A Tat subunit vaccine confers protective immunity against the immune-modulating activity of the human immunodeficiency virus type-1 Tat protein in mice.

The rational design of new therapies against HIV-1 necessitates an improved understanding of the mechanisms underlying the production of ineffective immune responses to HIV-1 in most infected individuals. This report shows that the CD8(+) T cell responses to gp120 were greatly diminished in mice vaccinated with a bicistronic gp120-Tat DNA vaccine, compared with those induced by a DNA vaccine encoding gp120 alone. The CD8(+) T cell responses induced by the latter included strong gp120-specific IFN-gamma secretion and protective antiviral immunity against challenge by a vaccinia-env pseudotype. The degree to which Tat influenced CD8(+) T cell responses depended on the bioactivity of Tat. Thus, a bicistronic DNA vaccine that expresses gp120 and a truncated Tat defective for LTR activation elicited strong IFN-gamma -secreting CD8(+) T cell responses to gp120 but conferred only marginal protection against the vaccinia-env challenge. The effect of Tat was completely blocked, however, by immunization with inactivated Tat protein before vaccination with the bicistronic gp120-Tat DNA vaccine.

An HIV-1 transgenic rat that develops HIV-related pathology and immunologic dysfunction.

We report, to our knowledge, the first HIV type 1 (HIV-1) transgenic (Tg) rat. Expression of the transgene, consisting of an HIV-1 provirus with a functional deletion of gag and pol, is regulated by the viral long terminal repeat. Spliced and unspliced viral transcripts were expressed in lymph nodes, thymus, liver, kidney, and spleen, suggesting that Tat and Rev are functional. Viral proteins were identified in spleen tissue sections by immunohistochemistry and gp120 was present in splenic macrophages, T and B cells, and in serum. Clinical signs included wasting, mild to severe skin lesions, opaque cataracts, neurological signs, and respiratory difficulty. Histopathology included a selective loss of splenocytes within the periarterial lymphoid sheath, increased apoptosis of endothelial cells and splenocytes, follicular hyperplasia of the spleen, lymphocyte depletion of mesenteric lymph nodes, interstitial pneumonia, psoriatic skin lesions, and neurological, cardiac, and renal pathologies. Immunologically, delayed-type hypersensitivity response to keyhole limpet hemocyanin was diminished. By contrast, Ab titers and proliferative response to recall antigen (keyhole limpet hemocyanin) were normal. The HIV-1 Tg rat thus has many similarities to humans infected with HIV-1 in expression of viral genes, immune-response alterations, and pathologies resulting from infection. The HIV-1 Tg rat may provide a valuable model for some of the pathogenic manifestations of chronic HIV-1 diseases and could be useful in testing therapeutic regimens targeted to stages of viral replication subsequent to proviral integration.

Toward a new generation of vaccines: the anti-cytokine therapeutic vaccines.

Pathological conditions, such as cancers, viral infections, and autoimmune diseases, are associated with abnormal cytokine production, and the morbidity associated with many medical disorders is often directly a result of cytokine production. Because of the absence of negative feedback control occurring in some pathophysiologic situations, a given cytokine may flood and accumulate in the extracellular compartment of tissues or tumors thereby impairing the cytokine network homeostasis and contributing to local pathogenesis. To evaluate whether the rise of anti-cytokine Abs by vaccination is an effective way to treat these pathological conditions without being harmful to the organism, we have analyzed each step of the cytokine process (involving cytokine production, target response, and feedback regulation) and have considered them in the local context of effector--target cell microenvironment and in the overall context of the macroenvironment of the immune system of the organism. In pathologic tissues, Abs of high affinity, as raised by anti-cytokine vaccination, should neutralize the pool of cytokines ectopically accumulated in the extracellular compartment, thus counteracting their pathogenic effects. In contrast, the same Abs should not interfere with cytokine processes occurring in normal tissues, because under physiologic conditions cytokine production by effector cells (induced by activation but controlled by negative feedback regulation) does not accumulate in the extracellular compartment. These concepts are consistent with results showing that following animal and human anti-cytokine vaccination, induction of high-affinity Abs has proven to be safe and effective and encourages this approach as a pioneering avenue of therapy.

Human papillomavirus-16-E7 oncoprotein enhances the expression of adhesion molecules in cervical endothelial cells but not in human umbilical vein endothelial cells.

OBJECTIVES: E7 is one of the oncoproteins encoded by human papillomavirus-16 (HPV-16), the major etiologic factor responsible for cervical cancer. Human papillomavirus-16-E7 expressed by human uterine cervix carcinoma cells is also released in the extracellular compartment where it induces immune suppression. We investigated whether E7 was also responsible for the enhanced endothelial adhesiveness required in cancer progression. STUDY DESIGN/METHODS: We treated cervical microvascular endothelial cells (CrMVEn) and human umbilical vein endothelial cells (HUVEC) with E7, tumor necrosis factor-alpha (TNF-alpha), and hydrogen peroxide (H2O2) and measured the expression of E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) by fluorescent-activated cell sorter analysis. RESULTS: E7 strongly induced the expression of E-selectin, ICAM-1, and VCAM-1 in CrMVEn, but not in HUVEC. Tumor necrosis factor-alpha further increased the endothelial expression of adhesion molecules in CrMVEn. Hydrogen peroxide pre-treatment resulted in an enhanced ICAM-1 and a decreased E-selectin and VCAM-1 expression. We also show indirect effects when endothelial cells were stimulated with the supernatant of E7-pretreated macrophages. CONCLUSIONS: These results show that HPV-16-E7 oncoprotein strongly induces adhesion molecules expression in organ-specific endothelial cells.

Perforin-low memory CD8+ cells are the predominant T cells in normal humans that synthesize the beta -chemokine macrophage inflammatory protein-1beta.

The synthesis of antiviral beta-chemokines has joined cytolysis as a potential mechanism for the control of HIV-1 infection by CD8(+) T cells. Recent evidence suggests that these two effector functions can diverge in some individuals infected with HIV-1; however, little is known about the CD8(+) T cell subsets in normal individuals that synthesize antiviral beta-chemokines. In this report, we have used mutliparameter flow cytometry to characterize the T cell subsets that secrete the antiviral beta-chemokine macrophage inflammatory protein (MIP)-1beta. These studies have shown: (i) CD8(+) cells are the predominant T cell subset that synthesizes MIP-1beta; (ii) MIP-1beta and IFN-gamma are synthesized congruently in most CD8(+) T cells; however, significant numbers of these cells synthesize only one of these effector molecules; (iii) approximately 60% of the CD8(+) T cells that synthesize MIP-1beta lack perforin; (iv) MIP-1beta is synthesized with approximately equal frequency by CD28(+) and CD28(-) subpopulations of CD8(+) T cells; (v) MIP-1beta is synthesized by three distinct CD8(+) T cell subsets defined by the expression of CD45R0 and CD62L; and (vi) MIP-1beta is not synthesized in short-term cultures of naive CD8(+) T cells. These results demonstrate substantial subset heterogeneity of MIP-1beta synthesis among CD8(+) T cells and suggest that these subsets should be evaluated as correlates of protective immunity against HIV-1.

Non-toxic immunogens for therapeutic anticytokine vaccines.

Over the last decade, the availability of purified cytokines and of cytokine antibodies (Abs) has prompted both scientists and pharmaceutical companies to develop anticytokine Ab therapy, and clinical trials have shown that anticytokine Abs are transiently effective. Active immunization may offer advantages over passive immunization in many situations. As anticipated from basic research, preclinical experiments and recent trials, such vaccines are safe and elicit high neutralizing anti-Ab titers. Repeated booster injections at 4 +/- 2 month intervals are however necessary to maintain long-term, high-affinity Ab response and efficacy. Anticytokine vaccines, alone or in combination with other therapies, represent potential treatments for chronic diseases such as AIDS, cancer, allergy and autoimmunity.

Some recent results on HIV pathogenesis with implications for therapy and vaccines.

In the third decade of AIDS research, we are still facing most of the challenges that emerged when the disease was characterized. While therapeutic approaches to control HIV infection are available in developed countries, novel principles to combat and prevent HIV infection are needed, due to the high cost and the negative effects of currently available drugs. In addition, it is now clear that this disease cannot be eliminated without a vaccine, which appears to be the only possible weapon to combat HIV in developing countries. We have focused on two approaches to fight HIV; one based on select members of a family of proteins, i.e. beta-chemokines, that we have show to interfere with HIV entry and replication, and another centered on a viral protein, HIV-1 Tat, that is crucially involved in HIV regulation and in some of the pathogenic manifestations of HIV infection. Studies from others and ourselves have shown that these two approaches are now ready to the leave experimental bench and move into clinical testing.

Pivotal role of cyclic nucleoside phosphodiesterase 4 in Tat-mediated CD4+ T cell hyperactivation and HIV type 1 replication.

We show here that HIV type 1 (HIV-1) Tat protein, in combination with anti-CD3/CD28 mAbs, promotes IL-2 production and proliferation of primary CD4(+) T lymphocytes, obtained from HIV-1-seronegative donors. This effect was observed when Tat was immobilized on a solid support, but it was not observed with soluble Tat. Such hyperactivation was accomplished by recruiting the rolipram-sensitive cyclic nucleoside phosphodiesterase 4 and resulted in increased susceptibility to HIV-1 infection. Accordingly, rolipram potently inhibited HIV-1 replication in cultures stimulated by anti-CD3/CD28 +/- Tat. These results add to the concept that decreasing Tat activity is an important addition to anti-HIV-1 therapy, and they suggest a target for anti-HIV-1 chemotherapy, phosphodiesterase 4.

Higher macrophage inflammatory protein (MIP)-1alpha and MIP-1beta levels from CD8+ T cells are associated with asymptomatic HIV-1 infection.

To test the hypothesis that beta-chemokine levels may be relevant to the control of HIV in vivo, we compared RANTES, MIP-1alpha, and MIP-1beta production from purified CD8(+) T cells from 81 HIV-infected subjects and from 28 uninfected donors. Asymptomatic HIV(+) subjects produced significantly higher levels of MIP-1alpha and MIP-1beta, but not RANTES, than uninfected donors or patients that progressed to AIDS. In contrast, beta chemokines in plasma were either nondetectable or showed no correlation with clinical status. The high beta-chemokine-mediated anti-HIV activity was against the macrophage tropic isolate HIV-1(BAL), with no demonstrable effect on the replication of the T-cell tropic HIV-1(IIIB). These findings suggest that constitutive beta-chemokine production may play an important role in the outcome of HIV-1 infection.

The path to the discoveries of human retroviruses.

Paul Ehrlich, gifted with knowledge, vision, and the capacity to bring his discoveries to practice, shaped the destiny of many biomedical scientific disciplines, including immunology, chemotherapy, hematology, cytology, and cancer research. His perceptive concept of receptors and ligands binding together in highly specific reactions was introduced just over a century ago in 1898 and is both fundamental and central to present day biomedical research. His mother country, Germany, commemorates his seminal contributions to science and to human welfare with an annual prize for achievements in fields that are related to his work. The 1999 Paul Ehrlich and Ludwig Darmsteaedter Prize was awarded to Robert C. Gallo, M.D. for his achievements in the pursuit of cancer related viruses and the growth in culture of human T-cells which led to the discovery of the first human retroviruses and, as a direct consequence, the discovery of the Human Immunodeficiency Virus, the third known human retrovirus. Below, excerpted from his acceptance lecture, is a concise personal history of those discoveries.

Vaccination with tat toxoid attenuates disease in simian/HIV-challenged macaques.

The Tat protein is essential for HIV type 1 (HIV-1) replication and may be an important virulence factor in vivo. We studied the role of Tat in viral pathogenesis by immunizing rhesus macaques with chemically inactivated Tat toxoid and challenging these animals by intrarectal inoculation with the simian/human immunodeficiency virus 89.6PD. Immune animals had significantly attenuated disease with lowered viral RNA, interferon-alpha, and chemokine receptor expression (CXCR4 and CCR5) on CD4(+) T cells; these features of infection have been linked to in vitro effects of Tat and respond similarly to extracellular Tat protein produced during infection. Immunization with Tat toxoid inhibits key steps in viral pathogenesis and should be included in therapeutic or preventive HIV-1 vaccines.

IFN-alpha 2b reduces IL-2 production and IL-2 receptor function in primary CD4+ T cells.

Initially described as an antiviral cytokine, IFN-alpha has been subsequently shown to affect several cellular functions, including cellular differentiation and proliferation. For these reasons, IFN-alpha is currently used in clinical practice for the treatment of viral infections and malignancies. In this manuscript, we show two novel mechanisms concomitantly responsible for the antiproliferative effect of IFN-alpha. First, long-term treatment with IFN-alpha of primary CD4+ T cells reduced surface expression of CD3 and CD28. These events resulted in decreased phosphorylation of the mitogen-activated extracellular signal-regulated activating kinase and its substrate extracellular signal-regulated kinase, leading to diminished production of IL-2. Second, IFN-alpha treatment of primary CD4+ T cells reduced proliferative response to stimulation in the presence of exogenous IL-2 by markedly decreasing mRNA synthesis and surface expression of CD25 (alpha-chain), a critical component of the IL-2R complex. These results may be relevant for the antitumor effects of IFN-alpha and may help us to better understand its detrimental role in the inhibition of proliferation of the bulk of CD4+ T cells (uninfected cells) in HIV-infected persons, who are known to overproduce IFN-alpha.

The role of chemokines in human immunodeficiency virus infection.

Chemokines that bind to human immunodeficiency virus (HIV) co-receptors are potent and selective inhibitors of HIV infection. Therefore, ever since our discovery of this activity, we have proposed their role in controlling HIV infection as a third arm of the immune response, i.e. in concert with humoral and cellular responses. Research carried out in our laboratory, and performed independently by other groups, has recently strengthened this concept. Here, we critically analyze the evidence indicating the positive contribution of chemokines to HIV infection, their involvement with cognate and innate immunity, and the potential for their use in combating HIV infection.