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Immunogenicity of a novel DNA vaccine cassette expressing multiple human immunodeficiency virus (HIV-1) accessory genes

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Immunogenicity of a novel DNA vaccine cassette expressing multiple human immunodeficiency virus (HIV-1) accessory genes

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Abstract

To develop an HIV-1 accessory gene immunogen using a DNA vaccine approach. HIV-1 accessory genes vif, vpu and nef were modified to express under the control of a single promoter with cellular proteolytic cleavage sites between the coding sequences (VVN-P). Immune responses induced by these constructs were evaluated in mice. DNA vaccine construct (pVVN-P) expressing Vif, Vpu and Nef was processed and the fusion protein was cleaved appropriately. Vif, Vpu and Nef as a fusion protein with proteolytic cleavage sites (VVN-P) is able to induce a significant level of cellular immune responses. We also observed that accessory genes Vif, Vpu and Nef (VVN-P) induced an effective T helper 1 proliferative response measured by cytokine production. Furthermore, expression cassette pVVN-P was able to induce cytotoxic T lymphocyte (CTL) responses against diverse HIV-1 viruses in infected target cells. We conclude that cell-mediated immune responses induced by accessory gene constructs from clade B may have a broader recognition of divergent HIV-1 viruses and should be further examined for both prophylactic and therapeutic vaccination schemes against HIV-1.

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... Consequently, immune containment of Nef functions could significantly contribute to controlling HIV infection . The other accessory proteins, Vpr, Vpu and Vif, which are expressed late in the virus life cycle, are also immunogenic30313233 and have been included in multicomponent candidate vaccine formulations to broaden virus-specific immunity313233. ...
... Consequently, immune containment of Nef functions could significantly contribute to controlling HIV infection . The other accessory proteins, Vpr, Vpu and Vif, which are expressed late in the virus life cycle, are also immunogenic30313233 and have been included in multicomponent candidate vaccine formulations to broaden virus-specific immunity313233. ...
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The HIV/AIDS epidemic has many serious consequences for children. These consequences are, however, rarely inevitable. Families can provide a protective barrier that deflects blows, or minimises their impact and a supportive nurturing environment that can help children recover from harm. If strong enough, and with sufficient access to quality services and support from communities, families can reduce the impacts of HIV/AIDS on children to negligible levels in most areas of impact. It is apparent that the impacts felt by children are not simply unfortunate, inevitable consequences of this epidemic. A strong and supported family with good access to quality services can deflect almost all of the impact. It is as a result of an interaction of the context of poverty, which weakens families, and a failure to adequately respond, that impacts are felt by children.
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The severity of the AIDS epidemic clearly emphasises the urgent need to expedite HIV vaccine candidates into clinical trials. Prophylactic HIV vaccine candidates have been evaluated in non-human primates. Based on specific proof of principle studies the first phase III clinical studies have recently begun in humans. However, a truly effective HIV vaccine is not yet at hand and many problems related to specific properties of the virus remain to be overcome. Previously proven empirical approaches have largely failed and now rational thinking based on an understanding of immunity to lentiviral infections is needed. This review addresses the scientific problems and complications facing the development of an HIV vaccine as well as the possible strategies currently available to overcome these problems. Recent attention has focussed on identifying the immune correlates and mechanisms of protection from either HIV infection or protection from disease progression. Based on these observations, the logic and rational behind the development of multiple component vaccine strategies are highlighted.
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Migration is very often a family affair, and often involves children, directly or indirectly. It may give rise to better quality of life for an entire family, or to bitter disappointment, and may also increase vulnerability to HIV and AIDS. This review, carried out for the Joint Learning Initiative on Children and AIDS, links the literature on “migration”, on “HIV and AIDS” and on “families”. Three themes are sketched: (1) As both HIV prevalence and circular migration increase, former migrant workers affected by AIDS may return to their families for care and support, especially at the end of life, often under crisis conditions. Families thus lose promising members, as well as sources of support. However, very little is known about the children of such migrants. (2) Following patterns of migration established for far different reasons, children may have to relocate to different places, sometimes over long distances, if their AIDS-affected parents can no longer care for them. They face the same adaptation challenges as other children who move, but complicated by loss of parent(s), AIDS stigma, and often poverty. (3) The issue of migrant families living with HIV has been studied to some extent, but mainly in developed countries with a long history of migration, and with little attention paid to the children in such families. Difficulties include involuntary separation from family members, isolation and lack of support, disclosure and planning for children's care should the parent(s) die and differences in treatment access within the same family. Numerous research and policy gaps are defined regarding the three themes, and a call is made for thinking about migration, families and AIDS to go beyond description to include resilience theory, and to go beyond prevention to include care.
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Two decades of HIV prevention efforts with men who have sex with men (MSM) have not eliminated the risk of new HIV infections in this vulnerable population. Indeed, current incidence rates in African American MSM are similar to those usually only seen in developing countries. A review of the existing literature suggests that the prevention research agenda for Black MSM could benefit from reframing conceptualization of risk as a function of individual properties to a broad consideration of social and interpersonal determinants. Studies that investigate dyadic and social-level influences on African American MSM's relationships are needed. This includes research explicating the diversity existing within the categorizations of Black MSM with respect to perceived identity (gay, bisexual, "men on the down low," "homo thugz"), constructions of masculinity, sexual scripts, sources of social support, and perceived norms and expectations. Recommendations are proposed for a research agenda focusing on linkages between interpersonal and social-structural determinants of HIV risk.
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This article reviews the current state of HIV/AIDS and youth research with special reference to South Africa. As a sector known to be at high risk for acquiring HIV infection, studies aimed at understanding young peoples’ vulnerability are a vitally important part of an informed response to the AIDS epidemic. Discussion focuses on the social context and contemporary sexual culture as shaping factors in the enactment of high‐risk sexual behaviour. Illustrating the importance of these factors, the author describes the rise of a particular sexual dynamic in one community in KwaZulu‐Natal, and analyses its implications for continued high rates of HIV transmission. The question emerges how best to focus research and inform efforts aimed at mediating the ‘dis‐enabling’ environments that effectively nullify safe‐sex messages.
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The Intervention with Microfinance for AIDS and Gender Equity (IMAGE) combines microfinance, gender/HIV training and community mobilization (CM) in South Africa. A trial found reduced intimate partner violence among clients but less evidence for impact on sexual behaviour among clients' households or communities. This process evaluation examined how feasible IMAGE was to deliver and how accessible and acceptable it was to intended beneficiaries during a trial and subsequent scale-up. Data came from attendance registers, financial records, observations, structured questionnaires (378) and focus group discussions and interviews (128) with clients and staff. Gender/HIV training and CM were managed initially by an academic unit ('linked' model) and later by the microfinance institution (MFI) ('parallel' model). Microfinance and gender/HIV training were feasible to deliver and accessible and acceptable to most clients. Though participation in CM was high for some clients, others experienced barriers to collective action, a finding which may help explain lack of intervention effects among household/community members. Delivery was feasible in the short term but both models were considered unsustainable in the longer term. A linked model involving a MFI and a non-academic partner agency may be more sustainable and is being tried. Feasible models for delivering microfinance and health promotion require further investigation.
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New outbreaks of human immunodeficiency virus type 1 (HIV-1) among injecting drug users (IDUs) are spreading in China along heroin trafficking routes. Recently, two separate HIV-1 epidemics among IDUs were reported in Guangxi, Southern China, where partial sequencing of the env gene showed subtype C and circulating recombinant form (CRF) AE. We evaluated five virtually full-length HIV-1 genome sequences from IDUs in Guangxi to determine the genetic diversity and the presence of intersubtype recombinants. Sequence analysis showed two geographically separated, highly homogeneous HIV-1 strains. B/C intersubtype recombinants were found in three IDUs from Baise City, in a mountainous region near the Yunnan-Guangxi border. These were mostly subtype C, with portions of the capsid and reverse transcriptase (RT) genes from subtype B. The subtype B portion of the capsid was located in the N-terminal domain, which has been shown to influence virus core maturation, virus infectivity, and binding to cyclophilin A, whereas the subtype B portion of RT was located in the palm subdomain, which is the active site of the enzyme. These BC recombinants differed from a BC recombinant found in Xinjiang Province in northwestern China. CRF AE strains were found in IDUs from Nanning, the capital of Guangxi, and in IDUs from Pingxiang City near the China-Vietnam border. The AE and BC recombinants were both remarkable for their low interpatient diversity, less than 1% for the full genome. Rapid spread of HIV-1 among IDUs may foster the emergence of highly homogeneous strains, including novel recombinants in regions with multiple subtypes.
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A decrease in HIV-related mortality and morbidity has been observed since 1996 in most developed countries as a consequence of the extensive use of combined antiretroviral therapies. The purpose of this study was to investigate whether combined antiretroviral therapies had a differential impact on the survival of patients with different AIDS-defining illnesses (ADIs). In total, 35,318 persons representing all the adults with AIDS (PWAs) diagnosed in Italy from January 1, 1990 to August 31, 1998 were studied. Actuarial life tables and the Kaplan-Meier method were used to estimate the cumulative probability of survival; the multivariate Cox proportional hazards model was used to estimate adjusted relative hazard of death (RH). Among PWAs diagnosed after 1995, the proportion of survivors 24 months after diagnosis was more than doubled (66%) compared with that of PWAs diagnosed before the end of 1995 (31%). Significantly decreased RHs for some ADIs were observed as early as 1996 (i.e., esophageal candidiasis, Pneumocystis carinii pneumonia, brain toxoplasmosis, HIV-wasting syndrome, and pulmonary tuberculosis). In the last period (1997-1998), the decrease was marked and significant for almost all the ADIs, ranging from 55% to 80% compared with the RHs of the reference year (1995). Conversely, primary lymphoma of the brain and Burkitt's lymphoma showed a low and not statistically significant decrease; these were the ADIs with the worst outcome. After 1995, there was a rather uniform increase in the survival of PWAs diagnosed with most specific ADIs but not for patients affected by primary brain lymphoma and Burkitt's lymphoma. The determinants of this differential effect need to be investigated.
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The drug treatments introduced in recent years for HIV infection have enabled a marked reduction in morbidity and prolongation of life. These treatments, however, are often associated with acute and chronic toxicities, the development of resistant virus can limit their effectiveness, and they are too expensive and difficult to administer in most third world settings. A successful HIV immunotherapeutic vaccine has the potential to overcome these problems, and would be a valuable advance. The most promising approaches have induced the type of immune response found to correlate with reduced activity of HIV in man, especially cytotoxic T-cell responses, or have led to reduced HIV or SIV viral load and increased CD4 counts in non-human primates or man. The agents that have led to one or both of these effects have been selected for review, and include inactivated envelope depleted virus, recombinant envelope glycoprotein, DNA vaccines utilising HIV peptides or gene products, viral vectors, such as canarypox or attenuated vaccinia, with HIV core proteins. There are other approaches, such as alloimmunity, for which no candidate products yet exist, but which conceptually appear promising. Currently, however, only a few phase III studies of HIV therapeutic vaccines have been completed in man, and there has been a modest therapeutic effect. Further development of both existing and new candidates remains one of the key priorities in our fight against HIV.
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The authors review ways in which laws and policies may affect individual and community HIV risk and resilience either by acting as pathways to disease or by structuring social determinants of disease. HIV serves as a rich case study. Increasingly HIV is concentrated among the marginalized who face prior stigma and discrimination and also bear a disproportionate burden of other diseases. To the degree that law and policy can alter the equation of vulnerability and of risk and resilience, they promise to be key public health interventions.
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Protease inhibitors (PI) produce significant immune recovery in most HIV-infected persons, although toxicity and high pill burden often limit this benefit. Combinations including non-nucleoside reverse transcriptase inhibitors (NNRTI) result in similar virological success, but data on immune reconstitution are scarce. Baseline plasma viraemia and CD4 cell counts were recorded from 100 patients who began two nucleoside analogue reverse transcriptase inhibitors plus either one PI or one NNRTI in a case-control study [indinavir (82%) and nevirapine (80%), respectively, were most frequently prescribed]. Only patients with baseline CD4 cell counts < 500 x 10(6) cells/l, good treatment adherence and plasma HIV RNA < 50 copies/ml sustained for 1 year were recruited. A rapid CD4 cell gain occurred within 12 weeks on therapy (average 41.8 x 10(6) cells/l per month), irrespective of treatment. In contrast, a trend towards a better CD4 cell gain was noticed between 12 and 48 weeks with a PI (mean CD4 cell increases per month: 15.2 x 10(6) cells/l using PI and 10.4 x 10(6) cells/l using NNRTI). During this period, the difference between therapy with a PI and a NNRTI reached statistical significance for subjects with baseline CD4 counts < 300 x 10(6) cells/l (17.1 x 10(6) and 6.4 x 10(6) cells/l, respectively; P < 0.05). A rapid CD4 cell increase occurred shortly after beginning antiretroviral therapy using either PI or NNRTI. Late increases in CD4 cell counts, mostly owing to newly produced cells rather than redistribution, are more pronounced in therapy using a PI, especially in subjects with lower initial CD4 cell counts.
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Since the first descriptions of HIV/AIDS HIV-1 has emerged as the most significant pathogen worldwide with approaching 60 million infections to the end of 2000. Over 18 million deaths from AIDS have occurred and HIV/AIDS is now classed among the top five causes of global mortality [1]. As with other infectious pathogens that exhibit a high degree of genetic variation such as influenza the extensive genetic variability of HIV-1 poses significant challenges for disease control and epidemiological surveillance [2]. The genetic heterogeneity of HIV-1 isolates is one of the major characteristics of the virus and the epidemic. This diversity generated by the processes of mutation and recombination [3] has led to the development of a subtype nomenclature for the classification of isolates [4]. The major (M) group of HIV-1 responsible for the majority of infections in the epidemic contains several recognized non-recombinant subtypes and sub-sub-types and at least 10 circulating recombinant forms (CRFs) [4]. These subtypes show uneven regional distribution and past attempts have been made to use them to describe the global molecular epidemiology of HIV-1 [56]. In this context undertaking systematic sampling and monitoring of populations is important to determine the value of these studies. We review here the public health and clinical significance of HIV-1 genetic variability and the methods and rationale for public health surveillance of subtypes. (excerpt)
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Viral drug susceptibility is associated with virologic response to new treatments. Standardized drug resistance tests are now available, and data from some clinical trials suggest that the use of drug resistance testing may be associated with improved virologic outcome. However, drug resistance testing is complex in terms of performance, interpretation and clinical application. HIV-1 drug resistance testing is used across Europe in patient management, but not in a consistent manner. This is due to differences in the national approaches to treatment, treatment management and reimbursement, as well as availability of tests. National guidelines only exist in some countries. In addition, the laboratory quality assurance and quality control standards are not applied uniformly. The EuroGuidelines Group was established to formulate clinical as well as laboratory guidelines for the use of HIV-1 drug resistance testing that are specific for the European setting. The group is comprised of academic clinicians and virologists, scientist from the industry and representatives of the patient community. The panel of experts will review these guidelines and update them on a yearly basis as new scientific evidence becomes available. (C) 2001 Lippincott Williams & Wilkins.
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At the present time, many AIDS clinical trials compare drug therapies by a time-to-event primary endpoint that measures the durability of suppression of HIV replication. For such studies, survival differences tend to occur early and/or late in the follow-up period due to drug differences in initial potency and/or durability of efficacy, and detecting these differences is of primary interest. We propose a weighted log-rank statistic that emphasizes early and/or late survival differences. We also consider some versatile tests that also emphasize these differences but are sensitive to a wider range of alternatives. The performances of the new tests are evaluated in numerical studies. For the alternatives of interest, the new tests show greater power and flexibility than commonly used weighted log-rank tests and related versatile tests. When the main interest is in detecting early and/or late survival differences, these tests may be preferable to the other versatile and weighted log-rank tests that have been studied.
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Over the prior 2 decades, HIV and AIDS have ravaged the Black community. This article summarizes the epidemiologic, social, and psychological impact of HIV/AIDS on individuals and affected subpopulations in the Black community. An overview is then provided of prevailing research on psychological and mental health issues in HIV/AIDS-related prevention and care that highlights key issues of concern to Black psychologists and areas in which Black psychologists are well positioned to make important contributions to the field. In conclusion, specific suggestions for Black psychologists to become more involved in work on HIV/AIDS are provided.
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Although cytotoxic T lymphocytes (CTLs) are thought to be involved in the control of human immunodeficiency virus-type 1 (HIV-1) infection, it has not been possible to demonstrate a direct relation between CTL activity and plasma RNA viral load. Human leukocyte antigen-peptide tetrameric complexes offer a specific means to directly quantitate circulating CTLs ex vivo. With the use of the tetrameric complexes, a significant inverse correlation was observed between HIV-specific CTL frequency and plasma RNA viral load. In contrast, no significant association was detected between the clearance rate of productively infected cells and frequency of HIV-specific CTLs. These data are consistent with a significant role for HIV-specific CTLs in the control of HIV infection and suggest a considerable cytopathic effect of the virus in vivo.
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The great variability of protein sequences from human immunodeficiency virus (HIV) type 1 (HIV-1) isolates represents a major obstacle to the development of an effective vaccine against this virus. The surface protein (Env), which is the predominant target of neutralizing antibodies, is particularly variable. Here we examine the impact of variability among different HIV-1 subtypes (clades) on cytotoxic T-lymphocyte (CTL) activities, the other major component of the antiviral immune response. CTLs are produced not only against Env but also against other structural proteins, as well as some regulatory proteins. The genetic subtypes of HIV-1 were determined for Env and Gag from several patients infected either in France or in Africa. The cross-reactivities of the CTLs were tested with target cells expressing selected proteins from HIV-1 isolates of clade A or B or from HIV type 2 isolates. All African patients were infected with viruses belonging to clade A for Env and for Gag, except for one patient who was infected with a clade A Env-clade G Gag recombinant virus. All patients infected in France were infected with clade B viruses. The CTL responses obtained from all the African and all the French individuals tested showed frequent cross-reactions with proteins of the heterologous clade. Epitopes conserved between the viruses of clades A and B appeared especially frequent in Gag p24, Gag p18, integrase, and the central region of Nef. Cross-reactivity also existed among Gag epitopes of clades A, B, and G, as shown by the results for the patient infected with the clade A Env-clade G Gag recombinant virus. These results show that CTLs raised against viral antigens from different clades are able to cross-react, emphasizing the possibility of obtaining cross-immunizations for this part of the immune response in vaccinated individuals.
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Lack of disease in long-term nonprogressors with human immunodeficiency virus type 1 (HIV-1) infection was strongly associated with very low copy numbers of HIV-1 DNA and RNA in peripheral blood mononuclear cells and plasma and the presence of high levels of anti-HIV-1 CD8+ memory cytotoxic T lymphocytes specific for Gag, Pol, and Env, compared with levels present in intermediate and advanced progressors. CD8+ memory cytotoxic T lymphocytes may have an important role in controlling HIV-1 replication and preventing disease in long-term nonprogressors.
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To gain more insight into the role of HIV-1-specific cytotoxic T lymphocytes (CTL) in the pathogenesis of AIDS, we investigated temporal relations between HIV-1 Gag-specific precursor CTL (CTLp), HIV-1 viral load, CD4+ T cell counts, and T cell function. Six HIV-1-infected subjects, who were asymptomatic for more than 8 yr with CD4+ counts > 500 cells/mm3, were compared with six subjects who progressed to AIDS within 5 yr after HIV-1 seroconversion. In the long-term asymptomatics, persistent HIV-1 Gag-specific CTL responses and very low numbers of HIV-1-infected CD4+ T cells coincided with normal and stable CD4+ counts and preserved CD3 mAb-induced T cell reactivity for more than 8 yr. In five out of six rapid progressors Gag-specific CTLp were also detected. However, early in infection the number of circulating HIV-1-infected CD4+ T cells increased despite strong and mounting Gag-specific CTL responses. During subsequent clinical progression to AIDS, loss of Gag-specific CTLp coincided with precipitating CD4+ counts and severe deterioration of T cell function. The possible relationships of HIV-1 Gag-specific CTLp to disease progression are discussed.
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We have been studying a patient who acquired human immunodeficiency virus (HIV) infection via a blood transfusion 13 years ago. She has remained asymptomatic since that time. The blood donor and two other recipients have all died of AIDS. Although this patient has shown persistently strong seroreactivity to HIV type 1 (HIV-1) antigens by Western blot (immunoblot), she has been continually HIV culture negative in results from multiple laboratories over the last 6 years and has a very low viral burden. Her CD4+ T-cell count has fluctuated around a mean of 399 cells per microliters, with little change in lymphocyte subset percentages. Strong cellular immune responses to HIV-1 epitopes by this patient have been demonstrated. We now report the results of an intensive molecular genetic analysis of the HIV-1 proviral quasispecies from this patient sampled over 5 years. Long terminal repeat region sequences supported the argument for normal basal and Tat-mediated promoter activities. Sequential sequencing of the nef gene revealed a low frequency (8.3%) of defective genes and a striking lack of sequence evolution. Functional analysis of predominant nef genes by both a cell surface CD4 downregulation and a viral infectivity complementation assay showed wild-type function. In contrast, sequential analysis of an amplicon containing the vif, vpr, vpu, tat1, and rev1 genes revealed the presence of inactivating mutations in 64% of the clones. These data suggest that this patient, initially infected with a virulent swarm of HIV-1, is presently infected with a more-attenuated viral quasispecies as a result of effective host immunity.
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Replication of vif-negative human immunodeficiency virus type 1 (HIV-1) is attenuated in certain cell lines and highly impaired in peripheral blood lymphocytes in vitro. To determine whether intact vif is positively selected during natural HIV-1 infection and to determine vif sequence variability, we employed PCR amplification, cloning, and sequencing to investigate the vif region of replicating virus in short-term-passage HIV-1 primary isolates from five asymptomatic individuals and from five persons with AIDS. A total of 46 vif clones were obtained and analyzed. Recombinant proviruses were constructed from selected vif clones from one patient and found to be fully infectious. We found that 38 of the 46 clones sequenced carried open vif reading frames and that there was a low degree of heterogeneity of vif genes within isolates from the same individual and among isolates from different donors. The cysteines previously found to be essential for vif protein function were conserved in all clones. A phylogenetic tree constructed from all available vif nucleotide sequences resulted in a virus grouping similar to those of gag and env. Direct sequencing of vif amplified by PCR from uncultured lymphocytes of 15 individuals at various stages of progression toward AIDS demonstrated vif open reading frames in 13 of 15 samples tested. There was no obvious correlation between disease status and the presence of an intact vif within this sample group at the time of sample procurement. The conservation of the vif open reading frame in vitro and in vivo and its limited variability following virus transmission in vitro are consistent with a role for vif in natural HIV-1 infection.
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Virologic and immunologic studies were performed on five patients presenting with primary human immunodeficiency virus type 1 (HIV-1) infection. CD8+ cytotoxic T lymphocyte (CTL) precursors specific for cells expressing antigens of HIV-1 Gag, Pol, and Env were detected at or within 3 weeks of presentation in four of the five patients and were detected in all five patients by 3 to 6 months after presentation. The one patient with an absent initial CTL response had prolonged symptoms, persistent viremia, and low CD4+ T-cell count. Neutralizing antibody activity was absent at the time of presentation in all five patients. These findings suggest that cellular immunity is involved in the initial control of virus replication in primary HIV-1 infection and indicate a role for CTL in protective immunity to HIV-1 in vivo.
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Recently, immunization techniques in which DNA constructs are introduced directly into mammalian tissue in vivo have been developed. In theory, gene inoculation should result in the production of antigenic proteins in a natural form in the immunized host. Here we present the use of such a technique for the inoculation of mice with a human immunodeficiency virus type 1 (HIV-1) envelope DNA construct (pM160). Mice were injected intramuscularly with pM160 and were subsequently analyzed for their anti-HIV envelope immune responses. Antisera collected from inoculated animals reacted with the recombinant HIV-1 envelope in ELISA and immunoprecipitation assays. The antisera also contained antibodies that were able to neutralize HIV-1 infection and inhibit HIV-1-mediated syncytium formation in vitro. Furthermore, splenic lymphocytes derived from pM160-inoculated animals demonstrated HIV-envelope-specific proliferative responses. The gene inoculation technique mimics features of vaccination with live attenuated viruses and, therefore, may ultimately prove useful in the rapid development of safe and efficacious vaccines as it provides for production of relevant antigen in vivo without the use of infectious agents.
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Mice immunized with the regulatory genes nef, rev, and tat from human immunodeficiency virus type 1 developed both humoral and cellular immune responses to the gene products Nef, Rev, and Tat. This study demonstrates that it is feasible to induce immune reactions to all of these regulatory gene products. Humoral responses were seen after DNA boosts, while potent T-cell proliferative responses were noted already after a single immunization. A Th1-directed immune response was demonstrated early after immunization. A 3- to 75-fold-stronger T-cell response was seen in animals receiving DNA epidermally compared to that in animals receiving intramuscular injections. Nef, Rev, and Tat putative B- and T-cell epitopes were clearly mapped by using peptides derived from the regulatory proteins and were similar to those which are detected in human immunodeficiency virus infection. Although immunization by the Nef, Rev, and Tat proteins raised high immunoglobulin G titers in serum, the epitope spreading appeared broader after DNA immunization. The combination of all of these regulatory genes together with two genes for structural proteins, the envelope and gag genes, demonstrated that a combined approach is feasible in that reactivities to all antigens persisted or were even augmented. No interference between plasmids was noted.
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The vpr gene product of human immunodeficiency virus type 1 (HIV-1) is a virion-associated protein that is essential for efficient viral replication in monocytes/macrophages. Vpr is primarily localized in the nucleus when expressed in the absence of other viral proteins. Vpr is packaged efficiently into viral particles through interactions with the p6 domain of the Gag precursor polyprotein p55gag. We developed a panel of expression vectors encoding Vpr molecules mutated in the amino-terminal helical domain, leucine-isoleucine (LR) domain, and carboxy-terminal domain to map the different functional domains and to define the interrelationships between virion incorporation, nuclear localization, cell cycle arrest, and differentiation functions of Vpr. We observed that substitution mutations in the N-terminal domain of Vpr impaired both nuclear localization and virion packaging, suggesting that the helical structure may play a vital role in modulating both of these biological properties. The LR domain was found to be involved in the nuclear localization of Vpr. In contrast, cell cycle arrest appears to be largely controlled by the C-terminal domain of Vpr. The LR and C-terminal domains do not appear to be essential for virion incorporation of Vpr. Interestingly, we found that two Vpr mutants harboring single amino acid substitutions (A30L and G75A) retained the ability to translocate to the nucleus but were impaired in the cell cycle arrest function. In contrast, mutation of Leu68 to Ser resulted in a protein that localizes in the cytoplasm while retaining the ability to arrest host cell proliferation. We speculate that the nuclear localization and cell cycle arrest functions of Vpr are not interrelated and that these functions are mediated by separable putative functional domains of Vpr.
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The HIV-1 accessory gene product Vpr can influence viral pathogenesis by affecting viral replication as well as host cell transcription and proliferation. We have investigated the effects of Vpr on host cell activation and confirm that it influences cellular proliferation. However, we have also found that Vpr modulates T-cell receptor (TCR)-triggered apoptosis in a manner similar to that of glucocorticoids. In the absence of TCR-mediated activation, Vpr induces apoptosis whereas in its presence, Vpr interrupts the expected induction of apoptosis. This regulation of apoptosis is linked to Vpr suppression of NF-kappa B activity via the induction of I kappa B, an inhibitor of NF-kappa B. Further, Vpr suppresses expression of IL-2, IL-10, IL-12, TNF alpha and IL-4, all of which are NF-kappa B-dependent. The effects of Vpr could be reversed by RU486. Our finding that Vpr can regulate NF-kappa B supports the hypothesis that some aspects of viral pathogenesis are the consequence of cell dysregulation by Vpr.
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Human immunodeficiency virus type–1 (HIV-1) manipulates fundamental host cell processes in sophisticated ways to achieve optimum replicative efficiency. Recent studies have provided new details on the molecular interactions of HIV-1 with its host cell. For example, HIV-1 encodes a protein that regulates transcriptional elongation by interacting with a cellular cyclin-dependent kinase, another that activates the specific nuclear export of viral RNA, and several others that affect the intracellular trafficking of viral and host cell proteins. Detailed analysis of the interplay between these viral proteins and normal cellular activities has provided new insights into central questions of virology and host cell biology.
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nef alleles derived from a large number of individuals infected with human immunodeficiency virus type 1 (HIV-1) were analyzed to investigate the frequency of disrupted nef genes and to elucidate whether specific amino acid substitutions in Nef are associated with different stages of disease. We confirm that deletions or gross abnormalities in nef are rarely present. However, a comparison of Nef consensus sequences derived from 41 long-term nonprogressors and from 50 individuals with progressive HIV-1 infection revealed that specific variations are associated with different stages of infection. Five amino acid variations in Nef (T15, N51, H102, L170, and E182) were more frequently observed among nonprogressors, while nine features (an additional N-terminal PxxP motif, A15, R39, T51, T157, C163, N169, Q170, and M182) were more frequently found in progressors. Strong correlations between the frequency of these variations in Nef and both the CD4(+)-cell count and the viral load were observed. Moreover, analysis of sequential samples obtained from two progressors revealed that several variations in Nef, which were more commonly observed in patients with low CD4(+)-T-cell counts, were detected only during or after progression to immunodeficiency. Our results indicate that sequence variations in Nef are associated with different stages of HIV-1 infection and suggest a link between nef gene function and the immune status of the infected individual.
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To gain more insight into the role of HIV-1-specific cytotoxic T lymphocytes (CTL) in the pathogenesis of AIDS, we investigated temporal relations between HIV-1 Gag-specific precursor CTL (CTLp), HIV-1 viral load, CD4+ T cell counts, and T cell function. Six HIV-1-infected subjects, who were asymptomatic for more than 8 yr with CD4+ counts > 500 cells/mm3, were compared with six subjects who progressed to AIDS within 5 yr after HIV-1 seroconversion. In the long-term asymptomatics, persistent HIV-1 Gag-specific CTL responses and very low numbers of HIV-1-infected CD4+ T cells coincided with normal and stable CD4+ counts and preserved CD3 mAb-induced T cell reactivity for more than 8 yr. In five out of six rapid progressors Gag-specific CTLp were also detected. However, early in infection the number of circulating HIV-1-infected CD4+ T cells increased despite strong and mounting Gag-specific CTL responses. During subsequent clinical progression to AIDS, loss of Gag-specific CTLp coincided with precipitating CD4+ counts and severe deterioration of T cell function. The possible relationships of HIV-1 Gag-specific CTLp to disease progression are discussed.
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Cytotoxic T lymphocytes (CTL) are present at high activities in adult patients infected with the human immunodeficiency virus (HIV). In this report, CTL effectors were identified in peripheral blood mononuclear cells (PBMC) of children born to HIV-1-infected mothers. These CTL killed HLA-matched HIV-1-infected H9 target cells or doubly transfected P815-A2-env, gag or nef mouse tumor cells, which expressed the viral antigens in association with HLA-A1/A3 or HLA-A2, respectively. HIV-1-specific CTL were detected early after birth (less than 2 months) and remained present during the asymptomatic phase of the infection. As in HIV-1-infected adults, HIV-specific CTL declined with disease progression. Surprisingly, HIV-1-specific CTL were detected in the PBMC of three children who subsequently became seronegative.
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Overlap extension represents a new approach to genetic engineering. Complementary oligodeoxyribonucleotide (oligo) primers and the polymerase chain reaction are used to generate two DNA fragments having overlapping ends. These fragments are combined in a subsequent 'fusion' reaction in which the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the 3' extension of the complementary strand. The resulting fusion product is amplified further by PCR. Specific alterations in the nucleotide (nt) sequence can be introduced by incorporating nucleotide changes into the overlapping oligo primers. Using this technique of site-directed mutagenesis, three variants of a mouse major histocompatibility complex class-I gene have been generated, cloned and analyzed. Screening of mutant clones revealed at least a 98% efficiency of mutagenesis. All clones sequenced contained the desired mutations, and a low frequency of random substitution estimated to occur at approx. 1 in 4000 nt was detected. This method represents a significant improvement over standard methods of site-directed mutagenesis because it is much faster, simpler and approaches 100% efficiency in the generation of mutant product.
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Article
Human immunodeficiency virus type 1 (HIV-1) Env-, Gag-, Pol-, Nef-, and Tat-specific cytotoxic T-lymphocyte (CTL) activities were quantitated temporally in five patients with symptomatic primary HIV-1 infection. A dominant CD8(+)-mediated, major histocompatibility complex class I-restricted CTL response to the HIV-1 envelope glycoprotein, gp160, was noted in four of the five patients studied. The level of HIV-1-specific CTL activity in the five patients paralleled the efficiency of control of primary viremia. Patients who mounted strong gp160-specific CTL responses showed rapid reduction of acute plasma viremia and antigenemia, while in contrast, primary viremia and antigenemia were poorly controlled in patients in whom virus-specific CTL activity was low or undetectable. These results suggest that HIV-1-specific CTL activity is a major component of the host immune response associated with the control of virus replication following primary HIV-1 infection and have important implications for the design of antiviral vaccines.
Article
Major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) are part of the cellular immune response to human persistent virus infections. Measurements of the frequency and specificity of human immunodeficiency virus type 1 (HIV-1)-specific CTL and their variation with time may indicate their relative importance in modulating the progression of HIV-1 infection. We have used limiting dilution analysis (LDA) to derive quantitative estimates of the frequency of HIV-1-specific CTL precursors in a cross-sectional study of 23 patients at different clinical stages of HIV-1 infection and to compare these with the frequency of CTL precursors specific for another persistent virus (Epstein-Barr virus [EBV]) in the same patients. Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with autologous HIV-1-infected lymphoblasts and assayed for cytotoxicity in 51Cr release assays against autologous and MHC-mismatched lymphoblastoid B cells infected with recombinant vaccinia viruses expressing the three HIV-1 structural gene products. The frequency of MHC-restricted precursors was high in asymptomatic HIV-1-infected patients (env-specific CTL precursors up to 73/10(6) PBMC; gag-specific CTL precursors up to 488/10(6) PBMC), although the relative frequency against the different structural gene products varied from patient to patient. The HIV-1-specific CTL precursor frequency was reduced in patients who had more severe (< 400/microliters) CD4+ lymphocyte depletion, while in the majority of such patients the frequency of CTL precursors against EBV was maintained at levels observed in healthy controls. Direct CTL activity in unstimulated PBMC was observed in three of nine patients but no correlation was found between the presence of an activated CTL response and the magnitude of the CTL response detected after stimulation in LDA. Thus, CTL precursors were detected against all three HIV-1 structural gene products in patients with CD4+ lymphocyte counts > 400/microliters, at frequencies that are high compared with those reported for other persistent viruses. A CTL response directed against multiple protein antigens of HIV-1 may protect the patient against epitope variation. The fact that the EBV-specific CTL precursor frequencies were maintained in advanced HIV-1 infection suggests that there may be selective impairment of the HIV-1-specific CTL response associated with disease progression.
Article
Macrophages and lymphocytes are the two main targets for productive HIV-1 infection in vivo. To compare the effects of the "nonessential" HIV-1 accessory genes vpr, vpu, and nef on viral replication in these primary cell types, we generated a panel of mutant viruses derived from a molecularly cloned macrophage-tropic HIV-1 primary isolate. Mutant viruses had markedly different patterns of replication in macrophages, in contrast to lymphocytes in which differences were modest. Loss of vpr or vpu reduced viral antigen production in macrophages by up to 1000-fold, while replication in lymphocytes was only marginally affected. Loss of nef did not affect lymphocyte infection, but decreased replication in macrophages to a small extent. Mutation of multiple accessory genes restricted replication in both cell types, but to a much greater extent in macrophages, and frequently resulted in nonproductive infection. The degree to which replication depended on intact accessory genes varied in macrophages from different donors. The essential functions of these accessory genes in HIV-1 infection may be related to their combined effects in facilitating productive infection of macrophages.
Article
Although vigorous activated and memory CTL have been associated with HIV-1 infection, data are lacking regarding the breadth of epitopes recognized in a given individual and the relationship to the viral quasispecies present in vivo. In this study we performed a detailed analysis of the HIV-1-specific CTL response in a seropositive person with documented HIV-1 infection of 15 yr duration, stable CD4 counts above 500 cells/ml, and viral load persistently below 500 molecules of RNA/ml of plasma. Epitope mapping studies revealed the presence of HLA class I-restricted CTL responses to six different epitopes in p17, p24, RT, Env, and Nef, which conferred broadly cross-reactive recognition of reported HIV-1 variants. Sequence analysis of autologous viruses revealed the absence of immune escape variants within five of the six epitopes. Despite consistently low viral RNA levels in plasma and viral DNA levels in PBMC, in vivo-activated circulating CTL were detected against three of the epitopes. Five of the six epitopes, including the three dominant epitopes, have been detected in persons with progressive disease, suggesting that nonprogressors may not target unique epitopes. This study demonstrates that HIV-1-specific CTL can be highly activated and broadly directed in the setting of an extremely low viral load, and that neither high viral load nor antigenic diversity is required for the generation of a multispecific CTL response. Although the detection of strong CTL responses, low viral load, and lack of immune escape are consistent with the hypothesis that CTL may contribute to lack of disease progression in this individual, the contribution of these responses to maintenance of the asymptomatic state remains to be determined.
Article
Recent studies support the importance of investigating a DNA vaccination approach for the immunologic control of HIV-1. In this regard, it may be important to specifically engineer immune responses in order to improve on first generation vaccine attempts. Especially for HIV, induction of cell-mediated immunity may be an important feature for any candidate vaccine. In an attempt to engineer in vivo the enhancement of cellular immune response and to direct Ag-dependent immune response from Th2 to Th1 type, we investigated the role of codelivery of genes for IL-12 and granulocyte-macrophage-CSF along with DNA vaccine formulations for HIV-1 Ag. We found that codelivery of IL-12 expression cassettes with DNA vaccines for HIV-1 in mice resulted in splenomegaly as well as a shift in the specific immune responses induced. The codelivery of IL-12 genes resulted in the reduction of specific Ab response, while the coinjection of granulocyte-macrophage-CSF genes resulted in the enhancement of specific Ab response. In addition, we observed a significant Ag-specific stimulation of T cells with codelivery of both cytokines. Most importantly, we observed a dramatic increase in specific CTL response from the group coimmunized with the HIV-1 DNA vaccine and IL-12 genes. This work demonstrates the power of DNA delivery in vivo for both the production of a new generation of more effective and targeted vaccines or immunotherapies as well as an analytic tool for the molecular dissection of the mechanisms of immune function.
Article
A fundamental goal of current strategies to develop an efficacious vaccine for AIDS is the elicitation of broadly reactive cytotoxic T lymphocyte (CTL) reactivities capable of destroying virally infected targets. Recent application of recombinant canarypox ALVAC/HIV-1 vectors as vaccine immunogens in HIV-1,-noninfected volunteers has produced CTL responses in a significant number of vaccinees. Using a newly developed targeting strategy, we examined the capacity of vaccine-induced CTL to lyse autologous targets infected with a diverse group of viral isolates. CTL derived from recipients of a canarypox ALVAC/HIV-1 gp160 (MN) vaccine were found capable of lysing autologous CD4+ lymphoblasts infected with the prototypic LAI strain of HIV-1. When tested against autologous targets infected with primary HIV-1 isolates representing genetically diverse viral clades, CTL from ALVAC/gp160 recipients showed both a broad pattern of cytolysis in which viruses from all clades tested were recognized as well as a highly restricted pattern in which no primary isolates, including clade B, were lysed. Differences in the HLA haplotypes of the volunteers immunized with the envelope vector might be a major determinant of the relative breadth of their CTL response. In contrast to ALVAC/gp160 vaccinees, recipients of the ALVAC/HIV-1 immunogen containing envelope as well as gag and protease genes consistently had CTL reactivities effective against a spectrum of primary isolate-infected targets. These studies demonstrate for the first time that clade B-based canarypox vaccines can elicit broad CTL reactivities capable of recognizing viruses belonging to genetically diverse HIV-1 clades. The results also reinforce the impact of viral core elements in the vaccine as well as the pattern of major histocompatibility complex class I allelic expression by the vaccine recipient in determining the relative breadth of the cellular response.
Article
The identification of HIV-1-infected individuals who remain asymptomatic despite prolonged infection presents a unique opportunity to understand virologic and host factors involved in the pathogenesis of AIDS. We have previously identified 10 long-term survivors (LTS) who are clinically healthy and immunologically normal despite 13 to 15 years of HIV-1 infection. In this study, we examined three accessory genes of HIV-1, vif, vpr, and vpu, in these LTS. A total of 52 vif, 54 vpr, and 55 vpu nucleotide sequences were obtained from the peripheral blood mononuclear cells of these patients. Analysis of these sequences revealed no gross deletions or insertions. Most of the clones were full-length with an intact open reading frame. Phylogenetic analyses of the vif, vpr, and vpu sequences from the LTS suggested that the HIV-1 strains found in the study subjects are not significantly different from those found in patients with AIDS and that the viruses in the LTS are unlikely to share a common genetic origin. Furthermore, a similar degree of overall genetic diversity between viruses from the LTS and AIDS patients suggests that there is unlikely a significant correlation between the degree of genetic diversity and the rate of disease development. Factors other than genetic divergence, such as viral load and phenotype, are likely to impact more on disease status.
Article
Several members of the chemokine-receptor family serve, in conjunction with CD4, as receptors for the entry of human immunodeficiency virus type I (HIV-1) into cells. The principal receptor for entry of macrophage-tropic (M-tropic) HIV-1 strains is CCR5, whereas that for T-cell-line-tropic (T-tropic) strains is CXCR4. Unlike HIV-1, infection with either M-tropic or T-tropic strains of simian immunodeficiency virus (SIV) can be mediated by CCR5, but not CXCR4. SIV strains will also infect CD4+ cells that lack CCR5, which suggests that these strains use as yet unidentified receptors. Here we use an expression-cloning strategy to identify SIV receptors and have isolated genes encoding two members of the seven-transmembrane G-protein-coupled receptor family that are used not only by SIVs, but also by strains of HIV-2 and M-tropic HIV-1. Both receptors are closely related to the chemokine-receptor family and are expressed in lymphoid tissues. One of the receptors is also expressed in colon and may therefore be important in viral transmission. Usage of these new receptors following experimental infection of non-human primates with SIV strains may provide important insight into viral transmission and the mechanisms of SIV- and HIV-induced acquired immune-deficiency syndrome.
Article
To develop a putative immunization cassette using HIV-1 vif accessory gene derived from HIV-1 clinical specimens as a component of a DNA vaccine for HIV-1. vif genes were cloned from HIV-1-infected patients and the sequence variation present within the patients was analyzed. Prototypic genetic variants were selected and the ability of these clones to induce humoral and cellular immune responses was studied in animals. The selected protective genetic variants were biologically characterized through transcomplementation assays using primary cells infected with a vif-defective HIV-1 proviral clone. Analysis of vif variants from different patients revealed that vif is highly conserved with the open reading frame remaining intact in vivo. It was shown that attenuated vif clones from HIV-1-infected subjects can effectively induce both humoral and cellular responses against Vif protein in mice. Evaluation of the cellular responses in vitro using human cellular targets infected with a clinical HIV-1 isolate showed that vif clones could induce cellular responses capable of destroying the virus. The vif variants developed in this study exhibited non-productive phenotypes, yet were capable of inducing specific immune responses against HIV-1. These constructs could be used as part of a DNA vaccine strategy for HIV-1. This vaccine adaptation strategy could be used for the development of immunogens for any pathogen resulting in cross-reactive immunity and attenuated gene pathogenesis.
Article
Much remains to be discovered about the many roles of the auxiliary proteins of HIV-1 in the viral life cycle, and the list of functions delineated above could well be incomplete. Nevertheless, clear progress in defining the mechanisms of action, and cellular targets, for several key auxiliary proteins has now been made. Surprisingly, as summarized in Table 1Table 1, it appears that a similar strategy has been adopted by several different auxiliary proteins to achieve a range of distinct biological activities. In particular, it appears that a common mode of action for several of these proteins is to act as a molecular connector between two macromolecules that would otherwise be unable to interact. By this simple expedient, these small proteins are each able to recruit entire cellular metabolic pathways to the cause of enhancing the efficiency of virus replication.Table 1Biological Activities of Several HIV-1 Auxilary ProteinsAuxiliary ProteinActivityMechanismTatTranscriptional activation of HIV-1 LTRRecruitment of cyclin T to TAR RNARevNuclear Export of late HIV-1 mRNAsRecruitment of Crm1 to RRE RNANefDown-regulation of cell surfaceRecruitment of AP-1 or AP-2 to CD4NefDown-regulation of cell surfaceRecruitment of AP-1 or AP-2 to MHC IVpuDegradation of CD4 in the ERRecruitment of h-βTrCP to CD4
Article
Expression of human immunodeficiency virus–type 1 (HIV-1) Vpr after productive infection of T cells induces cell cycle arrest in the G2 phase of the cell cycle. In the absence of de novo expression, HIV-1 Vpr packaged into virions still induced cell cycle arrest. Naturally noninfectious virus or virus rendered defective for infection by reverse transcriptase or protease inhibitors were capable of inducing Vpr-mediated cell cycle arrest. These results suggest a model whereby both infectious and noninfectious virions in vivo, such as those surrounding follicular dendritic cells, participate in immune suppression.
Article
Despite considerable advances in antiviral therapy for HIV infection, successful global intervention will require an effective vaccine. Current evidence suggests that cytotoxic T lymphocyte responses will be an important component of such vaccination. Recent evidence suggests that cytotoxic T lymphocytes raised against viral antigens from different clades (subtypes) of HIV.1 can cross-react extensively and such data have major implications for HIV vaccine design.
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