被膜蛋白糖基化在HIV感染中的作用

在HIV感染过程中,病毒被膜蛋白糖基化起着重要作用。它使病毒粒子具有高度糖基化的表面,帮助HIV逃避人体免疫细胞识别和攻击。在病毒入侵时,被膜糖蛋白与宿主细胞表面的受体结合,并进行一系列构象变化,使病毒粒子顺利地与宿主细胞膜融合。介绍近年来对被膜蛋白糖基化过程与HIV成熟、感染和逃避免疫应答等方面分子水平作用机理的深入了解,这些作用机理将会有助于艾滋病疫苗的研制和以“糖链为靶”药物的开发。     

Tumor necrosis factor-alpha inhibits the competence signal delivered by HIV to normal B cells

Polyclonal B cell activation is commonly observed in HIV-infected patients. The coordinate delivery of a number of signals is required for B cell response. This work was designed to better define the role of HIV in the first steps of normal human B cells activation. We show that the infectious virus or recombinant envelope proteins can render B cells responsive to the growth-promoting effect of several T cell-derived IL, IL-2, IL-4, and low m.w. (12-kDa) BCGF. HIV acts in the absence of monocytes and on different populations of B cells. The competence signal can be provided by recombinant gp160 envelope protein. CD4 molecule is not involved in the interaction of HIV with B cells. In addition, we demonstrate that tumor necrosis factor alpha has no promoting activity when B cells are preactivated by HIV and it can suppress the response of HIV-preactivated B cells to IL-2, IL-4, and 12-kDa BCGF. Thus, the HIV envelope can deliver an early signal to normal B cells and modulate B cell response to physiologic signals. The possible relevance of this phenomenon to the immune defect observed in HIV patients is discussed.     

Antigenic specificity of antibody-dependent cell-mediated cytotoxicity directed against human immunodeficiency virus in antibody-positive sera.

Antibody-dependent cell-mediated cytotoxicity (ADCC) specific for human immunodeficiency virus (HIV) has been described for HIV-infected individuals. To determine the antigenic specificity of this immune response and to define its relationship to the disease state, an ADCC assay was developed using Epstein-Barr virus-transformed lymphoblastoid cell line targets infected with vaccinia virus vectors expressing HIV proteins. The vaccinia virus vectors induced appropriate HIV proteins (envelope glycoproteins gp160, gp120, and gp41 or gag proteins p55, p40, p24, and p17) in infected lymphoblastoid cell lines as demonstrated by radioimmunoprecipitation and syncytia formation with c8166 cells. Killer cell-mediated, HIV-specific ADCC was found in sera from HIV-seropositive but not HIV-seronegative hemophiliacs. This HIV-specific response was directed against envelope glycoprotein but was completely absent against target cells expressing the HIV gag proteins. The ADCC directed against gp160 was present at serum dilutions up to 1/316,000. There was no correlation between serum ADCC titer and the stage of HIV-related illness as determined by T-helper-cell numbers. These experiments clearly implicated gp160 as the target antigen of HIV-specific ADCC activity following natural infection. Vaccines which stimulate antibodies directed against gp160, which are capable of mediating ADCC against infected cells, could be important for protection against infection by cell-associated virus.     

Limited breadth of a T-helper cell response to a human immunodeficiency virus envelope protein

Single-envelope human immunodeficiency virus (HIV) vaccines have been studied for more than a decade, with some successes in homologous challenge experiments in nonhuman primates but with no clear successes in clinical trials. To gain insight into the breadth of the immunity elicited by such vaccines, we have dissected the T-helper cell response of C57BL/6 mice to an individual, molecularly cloned envelope protein. Here, we report that T-helper cells responsive to HIV type 1 1035 envelope are very highly restricted in C57BL/6 animals: seven different hybridomas recovered from five separate mice recognized the same peptide, PKVSFEPIPIHYCAP, located in the C2 region of gp120. Three of these hybridomas were tested on a natural variant of the peptide but failed to respond. A more extensive analysis of whole splenic populations from other C57BL/6 mice immunized with the 1035 envelope reproducibly confirmed that the gp120-specific T-helper response was almost exclusively focused on a single epitope. We conclude that single-envelope vaccines may frequently fail to provoke an immune response sufficiently diverse to recognize variant sequences among circulating HIV. The results encourage the inclusion of more than one envelope in future vaccines to enhance the potential diversity and respective surveillance capacities of responding T-helper cell populations.     

Immune response to human immunodeficiency virus. In vivo administration of anti-idiotype induces an anti-gp160 response specific for a synthetic peptide

Anti-idiotypic antibodies (anti-Id) to chimpanzee antibodies directed against a synthetic peptide corresponding to a native epitope associated with gp41 of human immunodeficiency virus (HIV) envelope glycoprotein were produced in rabbits. The peptide was analogous to amino acid sequences 735 to 752 from the human T cell leukemia virus-IIIB isolate of HIV. Characteristics of the anti-Id preparation included: 1) detection of a shared determinant present on a second chimpanzee and one of three rabbit antibody preparations directed against the synthetic peptide, 2) failure to recognize an idiotype (Id) in BALB/c mouse antisera to the peptide, and 3) partial inhibition of the homologous chimpanzee Id preparation from binding either peptide or a recombinant HIV gp160 preparation. Immunization of BALB/c mice with the anti-Id induced an antipeptide response which bound a recombinant gp160 preparation without subsequent peptide or gp160 exposure. The anti-gp160 containing sera from mice immunized with anti-Id were able to inhibit the Id-anti-Id reaction indicating that an Id-positive antibody response was induced. This Id is not normally expressed in the murine anti-gp 160 immune response to the synthetic peptide and suggests that this anti-Id may activate normally silent clones. This study indicates that Id networks may be operational during the immune response to HIV epitopes. Alternatively, anti-Id may be useful in altering the serologic characteristics of an antibody response to HIV and may offer potential for modulating the immune response in this viral infection.     

A human immunodeficiency virus prime-boost immunization regimen in humans induces antibodies that show interclade cross-reactivity and neutralize several X4-, R5-, and dualtropic clade B and C primary isolates

A human immunodeficiency virus (HIV) vaccine that will be useful in diverse geographic regions will need to induce a broad immune response characterized by cross-clade immunity. To test whether a clade B-based HIV candidate vaccine could induce interclade humoral responses, including neutralizing activity against primary HIV-1 isolates, sera were tested from recipients of a vaccine consisting of recombinant canarypox virus vCP205 and recombinant gp120(SF2). Serum antibodies exhibited strong immunochemical cross-reactivity with V3 peptides from clades B, C, and F, with weaker activity for several V3 peptides from clades A, D, G, and H; essentially no reactivity could be demonstrated with V3 peptides from clades E and O. Extensive cross-clade reactivity was also documented by enzyme-linked immunosorbent assay with all nine recombinant HIV envelope glycoproteins tested from clades B, D, and E. In addition, vaccinees' sera displayed significant neutralizing activity against 5 of 14 primary isolates tested, including one X4 virus and two dualtropic viruses (from clade B) and two R5 viruses (from clades B and C). This is the first demonstration of the induction by a candidate HIV vaccine constructed from clade B laboratory strains of HIV of neutralizing activity against R5 and clade C primary isolates. The data suggest that, by virtue of their ability to induce cross-clade immune responses, appropriately formulated HIV vaccines based on a finite number of HIV isolates may ultimately be able to protect against the wide range of HIV isolates affecting the populations of many geographic regions.     

Induction of anti-HIV neutralizing antibodies by synthetic peptides.

Two synthetic peptides containing amino acid sequences analogous to the envelope glycoprotein of human T-lymphotropic virus (HTLV) type III (HTLV-III) and lymphadenopathy associated virus (LAV) were produced and used to immunize rabbits. The subsequent rabbit antisera neutralized HTLV-III infectivity in vitro. The two synthetic peptides corresponded to regions associated with the gp120 or gp41 subunits respectively, of human immunodeficiency virus (HIV). This data indicates that at least two neutralizing epitopes are present on the envelope glycoprotein of HIV and these epitopes are associated with two distinct virus envelope glycoproteins. Antisera generated against these peptides neutralized infectivity of two different isolates of HTLV-III. The data is discussed in terms of possible strategy for developing an effective vaccine against the etiologic agents of acquired immune deficiency syndrome (AIDS).     

Solid-phase proteoliposomes containing human immunodeficiency virus envelope glycoproteins

The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein gp120 mediates receptor binding and is the major target for neutralizing antibodies. A broadly neutralizing antibody response is likely to be a critical component of the immune response against HIV-1. Although antibodies against monomeric gp120 are readily elicited in immunized individuals, these antibodies are inefficient in neutralizing primary HIV-1 isolates. As a chronic pathogen, HIV-1 has evolved to avoid an optimal host response by a number of immune escape mechanisms. Monomeric gp120 that has dissociated from the functional trimer presents irrelevant epitopes that are not accessible on functional trimeric envelope glycoproteins. The resulting low level of antigenic cross-reactivity between monomeric gp120 and the functional spike may contribute to the inability of monomeric gp120 to elicit broadly neutralizing antibodies. Attempts to generate native, trimeric envelope glycoproteins as immunogens have been frustrated by both the lability of the gp120-gp41 interaction and the weak association between gp120 subunits. Here, we present solid-phase HIV-1 gp160DeltaCT (cytoplasmic tail-deleted) proteoliposomes (PLs) containing native, trimeric envelope glycoproteins in a physiologic membrane setting. We present data that indicate that the gp160DeltaCT glycoproteins on PLs are trimers and are recognized by several relevant conformational ligands in a manner similar to that for gp160DeltaCT oligomers expressed on the cell surface. The PLs represent a significant advance over present envelope glycoprotein formulations as candidate immunogens for HIV vaccine design and development.     

Induction of protective immunity to Friend murine leukemia virus in genetic nonresponders to virus envelope protein

(B10.A x A/WySn)F1, H-2a/a, mice are genetic nonresponders to the envelope protein of Friend murine leukemia helper virus (F-MuLV) when immunized with a recombinant vaccinia virus expressing F-MuLV env gene. In contrast these mice can be protectively immunized against leukemogenic Friend virus complex using formalin-fixed F-MuLV virions in CFA. To determine which viral proteins were responsible for this immune protection, virion proteins prepared by SDS-PAGE and electroelution were used to immunize mice. Purified gp70 envelope protein in CFA was capable of inducing strong immune protection against the challenge with Friend virus complex in H-2a/a mice. Immunologic studies demonstrated that immunized mice developed a virus-specific T cell proliferative response and showed IgM to IgG Ig class switching of virus-neutralizing antibodies. These results indicated that genetically controlled immune nonresponsiveness to F-MuLV envelope Ag in H-2a/a mice could be overcome using denatured viral envelope protein together with a strong adjuvant.     

HIV-1 Tat-based vaccines: from basic science to clinical trials

Vaccination against human immunodeficiency virus (HIV)-1 infection requires candidate antigen(s) (Ag) capable of inducing an effective, broad, and long-lasting immune response against HIV-1 despite mutation events leading to differences in virus clades. The HIV-1 Tat protein is more conserved than envelope proteins, is essential in the virus life cycle and is expressed very early upon virus entry. In addition, both humoral and cellular responses to Tat have been reported to correlate with a delayed progression to disease in both humans and monkeys. This suggested that Tat is an optimal target for vaccine development aimed at controlling virus replication and blocking disease onset. Here are reviewed the results of our studies including the effects of the Tat protein on monocyte-derived dendritic cells (MDDCs) that are key antigen-presenting cells (APCs), and the results from vaccination trials with both the Tat protein or tat DNA in monkeys. We provide evidence that the HIV-1 Tat protein is very efficiently taken up by MDDCs and promotes T helper (Th)-1 type immune responses against itself as well as other Ag. In addition, a Tat-based vaccine elicits an immune response capable of controlling primary infection of monkeys with the pathogenic SHIV89.6P at its early stages allowing the containment of virus spread. Based on these results and on data of Tat conservation and immune cross-recognition in field isolates from different clades, phase I clinical trials are being initiated in Italy for both preventive and therapeutic vaccination.     

Glycoprotein exchange vectors based on vesicular stomatitis virus allow effective boosting and generation of neutralizing antibodies to a primary isolate of human immunodeficiency virus type 1

Live recombinant vesicular stomatitis viruses (VSVs) expressing foreign antigens are highly effective vaccine vectors. However, these vectors induce high-titer neutralizing antibody directed at the single VSV glycoprotein (G), and this antibody alone can prevent reinfection and boosting with the same vector. To determine if efficient boosting could be achieved by changing the G protein of the vector, we have developed two new recombinant VSV vectors based on the VSV Indiana serotype but with the G protein gene replaced with G genes from two other VSV serotypes, New Jersey and Chandipura. These G protein exchange vectors grew to titers equivalent to wild-type VSV and induced similar neutralizing titers to themselves but no cross-neutralizing antibodies to the other two serotypes. The effectiveness of these recombinant VSV vectors was illustrated in experiments in which sequential boosting of mice with the three vectors, all encoding the same primary human immunodeficiency virus (HIV) envelope protein, gave a fourfold increase in antibody titer to an oligomeric HIV envelope compared with the response in animals receiving the same vector three times. In addition, only the animals boosted with the exchange vectors produced antibodies neutralizing the autologous HIV primary isolate. These VSV envelope exchange vectors have potential as vaccines in immunizations when boosting of immune responses may be essential.     

A hunter virus that targets both infected cells and HIV free virions: Implications for therapy

The design of ‘hunter’ viruses aimed at destroying human immunodeficiency virus (HIV) infected cells is an active area of research that has produced promising results in vitro. Hunters are designed to target exposed viral envelope proteins in the membranes of infected cells, but there is evidence that the hunter may also target envelope proteins of free HIV, inducing virus-virus fusion. In order to predict the effects of this fusion on therapy outcomes and determine whether fusion ability is advantageous for hunter virus design, we have constructed a model to account for the possibility of hunter-HIV fusion. The study was based on a target cell-limited model of HIV infection and it examined the hunter therapeutic effect on recovering the HIV main target cells, the activated CD4⁺ T lymphocytes. These cells assist in setting up an immune response to opportunistic infections. The study analyzed the hunter dual mechanisms to control infection and because of diverse estimates for viral production and clearance of HIV, simulations were examined at rates spanning an order of magnitude. Results indicate that without hunter-HIV fusion ability, hunters that kill HIV-infected cells lead to a substantial recovery of healthy cell population at both low and high HIV turnover rates. When hunter-HIV fusion is included, cell recovery was particularly enhanced at lower HIV turnover rates. This study shows that the fusion ability, in addition to hunter infection ability, could be a favorable attribute for improving the efficacy of hunter-viral therapy. These results provide support for the potential use of engineered viruses to control HIV and other viral infections.     

Acutely dysregulated, chronically disabled by the enemy within: T-cell responses to HIV-1 infection

Human immunodeficiency virus (HIV) infection causes chronic progressive immunodeficiency and immune dysregulaton. Although simple depletion of the major target of HIV infection, the CD4+ T cell, can explain much of the immunosuppression seen, there are multiple other factors contributing to the immune dysregulation. CD4+ T-cell depletion induces a range of homeostatic mechanisms that contribute to immune activation and cell turnover, providing a milieu conducive to further viral replication and cell destruction, resulting in functional defects in various lymphoid organs. These changes are progressive and in turn compromise the homeostatic processes. Further, the infection, like any other viral infection, provokes an active immune response consisting of both CD4+ and CD8+ T-cell responses. Both appear compromised, displaying aberrant memory cell production. While some of these defects result from viral variation and the chronicity of antigen presentation, other defects of memory cell production appear very early during the primary immune response limiting the viral specific T-cell responses from the outset. This, combined with the ability of the virus to escape any successful immune responses, results in an attenuated immune response that eventually becomes exhausted, characterized by progressive deficits in T-cell repertoire. Furthermore, negative regulatory mechanisms that normally control the immune response may be aberrantly invoked, perhaps directly by the virus, further compromising the efficacy of the immune response. Rational design of effective immunotherapies depends on a clear understanding of the processes compromising the immune response to HIV.     

Pathogenesis of human immunodeficiency virus infection.

The lentivirus human immunodeficiency virus (HIV) causes AIDS by interacting with a large number of different cells in the body and escaping the host immune response against it. HIV is transmitted primarily through blood and genital fluids and to newborn infants from infected mothers. The steps occurring in infection involve an interaction of HIV not only with the CD4 molecule on cells but also with other cellular receptors recently identified. Virus-cell fusion and HIV entry subsequently take place. Following virus infection, a variety of intracellular mechanisms determine the relative expression of viral regulatory and accessory genes leading to productive or latent infection. With CD4+ lymphocytes, HIV replication can cause syncytium formation and cell death; with other cells, such as macrophages, persistent infection can occur, creating reservoirs for the virus in many cells and tissues. HIV strains are highly heterogeneous, and certain biologic and serologic properties determined by specific genetic sequences can be linked to pathogenic pathways and resistance to the immune response. The host reaction against HIV, through neutralizing antibodies and particularly through strong cellular immune responses, can keep the virus suppressed for many years. Long-term survival appears to involve infection with a relatively low-virulence strain that remains sensitive to the immune response, particularly to control by CD8+ cell antiviral activity. Several therapeutic approaches have been attempted, and others are under investigation. Vaccine development has provided some encouraging results, but the observations indicate the major challenge of preventing infection by HIV. Ongoing research is necessary to find a solution to this devastating worldwide epidemic.     

Utility of human immunodeficiency virus type 1 envelope as a T-cell immunogen

Human immunodeficiency virus (HIV)-specific CD8 T lymphocytes are important for the control of viremia, but the relative utility of responses to the various HIV proteins is controversial. Immune responses that force escape mutations that exact a significant fitness cost from the mutating virus would help slow progression to AIDS. The HIV envelope (Env) protein is subject to both humoral and cellular immune responses, suggesting that multiple rounds of mutation are needed to facilitate viral escape. The Gag protein, however, has recently been shown to elicit a more effective CD8 T-cell immune response in humans. We studied 30 pigtail macaques for their CD8 T-lymphocyte responses to HIV-1 Env and simian immunodeficiency virus (SIV) Gag following prime/boost vaccination and intrarectal challenge with simian-human immunodeficiency virus SHIV_mn229. Eight CD8 Env-specific T-cell epitopes were identified and mapped in 10 animals. Animals that generated Env-specific CD8 T-cell responses had equivalent viral loads and only a modest advantage in retention of peripheral CD4 T lymphocytes compared to those animals without responses to Env. This contrasts with animals that generated CD8 T-cell responses to SIV Gag in the same trial, demonstrating superior control of viral load and a larger advantage in retention of peripheral CD4 T cells than Gag nonresponders. Mutational escape was common in Env but, in contrast to mutations in Gag, did not result in the rapid emergence of dominant escape motifs, suggesting modest selective pressure from Env-specific T cells. These results suggest that Env may have limited utility as a CD8 T-cell immunogen.     

In vivo protective anti-HIV immune responses in non-human primates through DNA immunization

Abstract: An effective immune response involves the specific recognition of and elimination of an infectious organism at multiple levels. In this context DNA immunization can present functional antigenic proteins to the host for recognition by all arms of the immune system, yet provides the opportunity to delete any genes of the infectious organism which code for antigens or pieces of antigens that may have deleterious effects. Our group has developed the use of nucleic acid immunization as a possible method of vaccination against Human immunodeficiency virus type 1 (HIV-1) [1,2,3,10,11,12]. Sera from non-human primates immunized with DNA vectors that express the envelope proteins from HIV-1 contain antibodies specific to the HIV-1 envelope. These sera also neutralize HIV-1 infection in vitro and inhibit cell to cell infection in tissue culture. Analysis of cellular responses is equally encouraging. T cell proliferation as well as cytotoxic T cell lysis of relevant env expressing target cells were observed. In addition, evidence that DNA vaccines are capable of inducing a protective response against live virus was demonstrated using a chimeric SIV/HIV (SHIV) challenge in vaccinated cynomologous macaques. We found that nucleic acid vaccination induced protection from challenge in one out of four immunized cynomolgus macaques and viral load was lower in the vaccinated group of animals versus the control group of animals. These data encouraged us to analyze this vaccination technique in chimpanzees, the most closely related animal species to man. We observed the induction of both cellular and humoral immune responses with a DNA vaccine in chimpanzees. These studies demonstrate the utility of this technology to induce relevant immune responses in primates which may ultimately lead to effective vaccines.     

Microdevices for examining immunological responses of single cells to HIV

More than 60 million people in the world have been diagnosed with HIV infections since the virus was recognized as the causative agent of AIDS in the 1980s. Even though more than half of the infected patients have died, effective disease treatment and prevention measures have not been established. ART (antiretroviral therapy) is the only proven HIV treatment that sustains the suppression of patient viraemia. Current routine approaches to treat HIV infections are targeted at developing vaccines that will induce humoral or cell memory immune responses. However, developing an effective vaccine has been challenging because the HIV mutates rapidly, which allows the virus to evade immune surveillances established against the previous strain. In addition, the virus is able to quickly establish a reservoir and treatment is difficult because of the general lack of knowledge about HIV immune response mechanisms. This review introduces common disease symptoms and the progression of HIV infection with a brief summary of the current treatment approaches. Different cellular immune responses against HIV are also discussed, with emphasis on a nanotechnology research that has focused on probing T-cell response to HIV infection. Furthermore, we discuss recent noteworthy nanotechnology updates on T-cell response screening that is focused on HIV infection. Finally, we review potential future treatment strategies based on the correlations between T-cell response and HIV infection.     

Sequence divergence and open regions of RNA secondary structures in the envelope regions of the 17 human immunodeficiency virus isolates.

Genetic variation during the course of infection of an individual is a remarkable feature of the acquired immune deficiency syndrome (AIDS) disease. This variation has been studied for the envelope protein encoding regions of seventeen different sequences from various isolates of human immunodeficiency virus (HIV) using multiple sequence comparison and calculation of variability. The open regions with little intramolecular base pairing in these envelope sequences are predicted by a recently developed statistical method. The minimum length L for a run of hypervariable sites, conserved sites, or open regions that gives significance at the 1% (or 0.1%) level is then determined by a scan statistical method. The results show that significant clusters of open regions predicted at the RNA levels correlate with significant clusters of hypervariable sites in the HIV envelope gene. Those significant genomic variations in HIVs seem to be manifested mainly in the extracellular portion of the envelope protein. Twelve potential antigenic determinants are predicted using an antigenic index method. Interestingly, the majority of the significant hypervariable regions in the exterior envelope protein (gp120) were predicted potential epitopes.