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A vaccine consisting of DNA priming followed by recombinant modified vaccinia Ankara (rMVA) boosting has achieved long-term control of a pathogenic challenge with a chimera of simian and human immunodeficiency viruses (SHIV-89.6P) in rhesus macaques. Based on these results, clade B HIV-1 DNA and rMVA immunogens have been developed for trials in humans. We conducted a first-time in humans phase I safety trial using the pGA2/JS2 (JS2) HIV-1 DNA priming vector expressing Gag, Pol, Env, Tat, Rev, and Vpu. Thirty HIV-uninfected adults were vaccinated with 0.3 or 3 mg of JS2 DNA, or a saline placebo, by intramuscular injection at months 0 and 2. Both doses of DNA were safe and well-tolerated with no differences between the control, 0.3 mg, or 3 mg groups (n = 6, 12, and 12, respectively) through 12 months of postvaccination follow- up. A chromium-release assay using fresh peripheral blood mononuclear cells (PBMCs) and a validated IFN-gamma ELISpot assay with frozen PBMCs failed to detect CD4(+) or CD8(+) HIV-1-specific T cell responses. HIV-specific neutralizing antibodies were also not detected. The vaccine is being further developed as a priming vector for a combined DNA plus rMVA prime/boost HIV vaccination regimen.

The thymus of HIV-seropositive patients can enlarge as CD4+ T cell counts increase on highly active anti-retroviral therapy (HAART). This may indicate development of new T cells or represent mature peripheral T cells recirculating to the thymus. To define the etiology of the enlargement, the thymuses of two HIV-infected individuals on HAART were biopsied. For more than 3 years before initiation of HAART, both patients (38 and 41 years of age) had documented CD4+ T lymphopenia. Peripheral blood samples were obtained to assess circulating CD4+ CD45RA+ CD62L+ T cells, which were thought to have recently developed in the thymus. Peripheral blood T cells from both patients and thymocytes from the second patient were also tested for levels of DNA episomes formed during T cell receptor gene rearrangement (T cell receptor rearrangement excision circles, TRECs). With HAART, peripheral blood CD4+ T cell counts increased from approximately 60/mm(3) to 552/mm(3) and 750/mm(3) for patients 1 and 2, respectively. Thymic biopsies from both patients showed normal thymus histology with active thymopoiesis. Percentages of peripheral blood CD4+ CD45RA+ CD62L+ T cells and quantitation of T cell TRECs also reflected active thymopoiesis in both patients. Thus, in these two HIV-seropositive adults examined after initiation of HAART, thymic enlargement represented active thymopoiesis. Thymopoiesis in adult AIDS patients may contribute to immune reconstitution even after prolonged CD4+ T lymphopenia.

The immunologic and virologic factors that impact on the rate of disease progression after acute infection with human immunodeficiency virus (HIV) type 1 are poorly understood. A patient with an extraordinarily rapid disease course leading to AIDS-associated death within 6 months of infection was studied intensively for the presence of anti-HIV immune reactivities as well as changes in the genetic and biologic properties of virus isolates. Although altered humoral responses were evident, the most distinctive immunologic feature was a nearly complete absence of detectable HIV-specific CTL responses. In addition to a rapid decline in CD3+CD4+ cells, elevated percentages of CD8+CD45RA+ and CD8+CD57+ cells and diminished CD8+CD45R0+ and CD8+CD28+ cells were evident. Primary viral isolates recovered throughout the course of infection exhibited limited sequence diversity. Cloned viral envelopes were found to have unusually broad patterns of coreceptor usage for cell-cell fusion, although infectivity studies yielded no evidence of infection via these alternative receptors. The infectivity studies demonstrated that these isolates and their envelopes maintained an R5 phenotype throughout the course of disease. The absence of demonstrable anti-HIV CTL reactivities, coupled with a protracted course of seroconversion, highlights the importance of robust HIV-specific immune responses in the control of disease progression.

One of the fundamental goals of current strategies to develop an efficacious vaccine for AIDS is the elicitation of cytotoxic T lymphocyte (CTL) reactivities capable of recognizing cells infected with different subtypes of the human immunodeficiency virus type 1 (HIV-1). In efforts to explore new vaccine candidates by the UNAIDS/WHO Vaccine Committee, we review the most recent data concerning CTL epitopes that are conserved among the different HIV-1 subtypes. Moreover, we examine HLA allelic frequencies in several different populations, to determine those that could contribute to the goal of a cumulative phenotype frequency (CP) of at least 80%. By analyzing conserved epitopes in the context of HLA restricting alleles, we define a set of HIV-1 gene regions that may have the greatest potential to induce cross-clade reactive CTLs. The absence of well-defined correlates of immune protection that link CTL epitopes to delayed disease progression and/or prevention of infection does not permit an assignment of rank order of the most relevant component of a candidate vaccine. Thus far, most of the studies conducted in clade B-infected patients to define conserved and immunodominant epitopes indicate gag and pol gene products to be the most conserved among the HIV-1 subtypes. Moreover, anti-Pol and -Gag CTL responses appear to correlate inversely with disease progression, suggesting that they should be among the first choice of antigens to be included in a candidate vaccine construct aimed at induction of broad CTL responses. The impact of a clade B-based vaccine as a worldwide candidate capable of inducing protective immune responses can be determined only after "in vivo" studies. Meanwhile, extensive parallel studies in populations infected with non-clade B HIV-1 subtypes should define the patterns of immunodominant epitopes and HLA for comparison with the data already collected in clade B-infected subjects.

The purpose of this study was to determine whether thymic transplantation in addition to highly active antiretroviral therapy (HAART) will restore T cell function in HIV infection. Eight treatment-naive HIV-infected patients with CD4+ T cell counts of 200-500/mm3 were randomized into thymic transplantation and control arms. All patients received HAART (zidovudine, lamivudine, and ritonavir) for 6 weeks prior to transplantation. Thymic transplantation was done without immunosuppression, using postnatal HLA-unmatched cultured allogeneic thymus tissue. Patients were immunized every 6 months with the neoantigen keyhole limpet hemocyanin (KLH) and the recall antigen tetanus toxoid (TT). T cell phenotype and function and T cell receptor rearrangement excision circles (TRECs) were assessed. Thymic allografts were biopsied at 2 months. Six HIV-infected patients completed the study. Four patients received cultured allogeneic postnatal thymic grafts, two others were controls. CD4+ T cell counts increased and T cell-proliferative responses to Candida antigen and TT normalized in all patients. Proliferative responses to KLH developed in three of four transplant recipients and one of two controls. Patients responding to KLH after secondary immunization had greater TREC increases compared with the patients who did not respond. All thymic allografts were rejected within 2 months. In summary, four of six patients developed T cell-proliferative responses to the neoantigen KLH over the first 2 years of HAART. The transplanted thymus tissue, however, was rejected. There was no clear difference in restoration of T cell function in the transplant recipients compared with the controls. Increases in TRECs after initiation of HAART may correlate with improved immune function.

CD8+ T lymphocytes from HIV+ individuals can potently suppress HIV-1 replication in a noncytolytic manner. This suppression appears to be multifactorial and the molecules contributing have not been fully elucidated. As an approach to this question we used herpesvirus saimiri (HVS) to transform CD8+ T lymphocytes from an HIV+ asymptomatic donor to a continuously growing, activation-independent, IL-2-dependent phenotype. The transformed cell population, termed CD8(HVS), had an activated phenotype, contained HVS sequences, did not shed infectious HVS virus, and was polyclonal. The CD8(HVS) cells, despite the absence of detectable CTL activity, potently suppressed HIV-1 production by both autologous and heterologous CD4+ cells from infected donors. The CD8(HVS) cells in coculture also suppressed virus production from PBMCs acutely infected with syncytium-inducing (SI) strains or NSI primary isolates of HIV-1. The supernatants from the CD8(HVS) cells and their concentrates derived from these supernatants were suppressive to NSI primary isolates of HIV-1 but not to SI strains. Fractionation of these concentrates showed that the suppressive activity was associated with low molecular mass (6500- to 19,300-Da) protein species. Western blotting and ELISA indicated that the CC chemokines MIP-1alpha, MIP-1beta, and RANTES were present in these fractions. Antibody-blocking studies with antibodies to the CC chemokines indicated that a significant portion of the soluble HIV-suppressive activity was due to these molecules. However, these experiments also suggested the inhibitory activity of the CD8(HVS) cells in coculture is not due exclusively to the CC chemokines. The HVS-transformed cells provide a useful tool for the study of noncytolytic CD8+ T lymphocyte-mediated suppression of HIV-1.

We investigated the safety and immunogenicity of a candidate HIV-1 vaccine, Env 2-3 (Chiron Biocine Co.), in combination with an adjuvant emulsion, MF59, with or without an additional immune modulator, MTP-PE 78 healthy HIV-1-seronegative adults. Sixteen subjects participated in a dose escalation study of MTP-PE in MF59 without Env 2-3, given at 0 and 1 months; 48 subjects participated in a study of a fixed dose of 30 micrograms of Env 2-3 in MF59 with increasing doses of MTP-PE (0, 5, 10, 25, 50, and 100 micrograms), and 14 subjects participated in a study of 100 micrograms of Env 2-3 in MF59 without MTP-PE. Subjects were assigned to study groups under a randomized, double-blind allocation. Subjects received immunization at 0, 1, and 6 months, and had the option of receiving a fourth dose at 12-18 months. Env 2-3 in MTP-PE/MF59 was associated with significant reactogenicity, in that severe, although self-limited systemic and/or local reactions occurred in 15 of 30 vaccinees. In contrast, Env 2-3 in MF59 without MTP-PE was relatively well tolerated, and severe local and/or systemic reactions occurred in only 2 of 18 subjects. Env 2-3 stimulated serum antibodies to HIV-1 envelope protein (gp120) as detected by Western blot in 39 of 43 subjects and to HIV-1 virus lysate by EIA in 28 of 43 subjects after three injections. The majority of subjects also developed EIA antibodies to recombinant gp120 (SF-2), gp120 (LAI), and V3 peptide (SF-2). Neutralizing antibodies to the homologous SF-2 strain developed in 30 of 43 and 27 of 34 subjects, and fusion inhibition antibodies in 25 of 43 and 15 of 36 subjects after three and four injections, respectively. Lymphoproliferative responses to the immunogen, Env 2-3 were observed in over 80% of the vaccinees examined, and CD4+ cytotoxic T cell activity directed against HIV-1 was noted transiently in 2 of 20 vaccinees. Addition of MTP-PE to Env 2-3 or increasing the dose of Env 2-3 from 30 to 100 micrograms did not augment immunogenicity. Env 2-3 in MF59 was well tolerated and immunogenic in HIV-1-seronegative individuals. The addition of MTP-PE significantly increased reactogenicity, but had little, if any, effect on immunogenicity.

We have studied a woman with transfusion-acquired HIV who appears to have contained infectious virus to consistently undetectable levels over a 13-year period without antiviral treatment. She received the infected transfusion for intra- and postpartum blood loss immediately after delivery of her second child in 1981. She had no acute febrile syndrome and has never had HIV-associated clinical signs or symptoms in the 13 years since infection. She was first tested and found positive for HIV antibodies in 1985, and the infected blood donor was diagnosed with AIDS in 1986 and died of AIDS-related complications in 1989. Two other recipients of packed erythrocytes from this donor (in 1980 and 1982) also became infected and were subsequently diagnosed with AIDS. Between January 1986 and April 1994, in the setting of continuous and unambiguous Western blot HIV-specific antibodies and intermittently positive low-level HIV DNA signal after polymerase chain reaction (PCR) amplification, more than 30 separate cell cocultures performed in several independent laboratories failed to yield evidence of infectious virus, despite special efforts to induce and detect HIV replication. Immunologically, a strong in vitro proliferative response to HIV envelope proteins also distinguished this subject from other asymptomatic HIV+ individuals.

Studies have shown that cytolytic T lymphocyte (CTL) responses may be critical to the clearance of the early viremia in acute HIV-1 infection. It is likely that these cells play an important role in prolonging the asymptomatic phase of the infection. Although HIV-1-specific CTL activity can be detected in direct assays of freshly isolated peripheral blood lymphocytes (PBL) from some infected individuals, this method fails to detect CTL that are present at low frequency and resting, memory CTL. For these reasons, direct CTL assays on PBL from seropositive individuals may underestimate the level of CTL immunity. As part of ongoing investigations of CTL activity in HIV-1-infected individuals, we developed a novel strategy for the detection and ex vivo expansion of HIV-1-specific CTL. This technique involves selective stimulation of PBL from seropositive individuals with autologous Epstein-Barr virus (EBV)-transformed, B-lymphoblastoid cell lines (B-LCL) infected with vaccinia vectors expressing various HIV-1 genes. Prior to their use for in vitro stimulation, B-LCL are treated with psoralen and UV light to inactivate vaccinia virus. After 1 week of stimulation, CTL activity in stimulated cultures is measured in a standard 51Cr release assay. This ex vivo expansion method can selectively increase the bulk culture CTL activity against env, gag and nef, even in some seropositive individuals with low CD4 counts and little evidence of HIV-1-specific CTL in assays of freshly isolated PBL. These expanded CTL are predominantly of the CD8+ phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

HIV-1 infection evokes a vigorous antiviral response that may participate in resolving the initial peak of plasma viremia and maintenance of the asymptomatic state. CD8+ T lymphocytes of HIV-1-infected individuals play a critical role in the cellular anti-HIV response. In agreement with previous reports, we observed a potent suppressive effect on HIV-1 production from autologous CD4+ T lymphocytes by CD8+ T lymphocytes from asymptomatic HIV-1-infected individuals. To elucidate the mechanism(s) of the nonlytic suppressive antiviral activity, we examined the effect of CD8+ T lymphocytes on the transcriptional activity of the HIV-1 promoter (HIV-LTR). CD8+ lymphocytes from HIV-1-infected asymptomatic individuals suppressed tat-mediated HIV-LTR transcription in CD4+ lymphocytes. HIV-LTR transcriptional activity was suppressed by CD8 lymphocytes to an extent similar to tat-mediated transcription whereas CMV immediate early gene promoter activity was not affected. In contrast to the suppressive effect seen with CD8+ lymphocytes from HIV-1-infected individuals, CD8+ lymphocytes from uninfected individuals did not significantly inhibit tat-mediated or HIV-LTR transcription. The transcriptional inhibitory activity was not MHC class I restricted and could be mediated by a soluble factor(s). Supernatants from some CD8+ T lymphocyte cultures from HIV-1+ individuals exerted an inhibitory effect on tat-mediated HIV-LTR transcription comparable to that seen with CD8+ cells. In conclusion, CD8+ lymphocytes from asymptomatic HIV-1+ individuals could suppress virus production by inhibiting HIV-1 gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

As a means of assessing the immunological impact of HIV infection in the chimpanzee, as well as the participation of the cellular components in the control of HIV infection in these animals, various aspects of cellular immunity were investigated in chronically HIV-infected chimpanzees. Eight HIV-1-infected chimpanzees were included in this study; two of them were infected for more than 5 years and six for nearly 3 years at the time of study. All of the chimpanzees received either 40 or 100 TCID50 of HTLV-IIIB. Circulating peripheral blood lymphocytes were studied by flow cytofluorimetric analysis in order to reveal possible alterations in the CD4:CD8 ratio, as well as in specific CD4+ and CD8+ cell subpopulations. Chronically infected chimpanzees did not present significant alterations in the percentage of CD4+ or CD8+ lymphocyte subsets. Interestingly, the CD8+/CD57+ cell subset was not detectable. The expression of markers for activation on circulating lymphocytes, usually higher in the HIV-infected patients, was not altered in infected animals. The functional aspects of specific anti-HIV-1 non-MHC and MHC-restricted cellular cytotoxic reactivities were also investigated. The results were compared with the findings in normal uninfected chimpanzees and in HIV-infected humans. Only one chimpanzee (881) developed a detectable, specific non-MHC-restricted anti-HIV-1- reactivity. Compared to that seen in humans, the ontogeny of this activity is delayed. Among the other infected chimpanzees, no specific anti-HIV cellular reactivities were detectable in the peripheral blood. In chimpanzees, HIV-1 infection evidently does not elicit the same strong cellular reactivity as that detected in infected patients. The absence of chronic cellular activation, despite continued viral replication, may highlight a key determinant in HIV-1-induced pathogenesis that is likewise absent in infected chimpanzees.

More than 200 sera from macaques immunized with several different vaccine preparations were tested in various assays with cells of human and macaque origin. Only in instances where whole inactivated SIV preparations were used for immunization, were reactivities found with normal human cells, and this was the case in every instance. Such sera produced a marked clumping of several normal human cell lines and exhibited strong staining of the cell surface in FACS analysis. In the presence of SIVDeltaB670, these sera also enhanced infectivity and fusion formation. When similar tests were performed with macaque cells as targets, such phenomena were not easily discernible. Likewise, there was no trace of such activities in sera from normal animals, animals chronically infected with SIV, or in those from animals which received recombinant viral subunits as vaccines. Finally, we show that in several instances where whole inactivated virus was used as a vaccine, there is a strong correlation between the titer of anticellular activity with protection.

Sensitive and reproducible assays for SIV infection and syncytium formation have been developed in which high titers of neutralizing and fusion-inhibiting antibodies can be recorded. These assays will contribute toward our understanding of the role of humoral responses in SIV vaccine strategies.

We have developed a series of murine monoclonal antibodies to a region of the 120 kD envelope glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1). This region has previously been implicated as a site for virus neutralization by antisera raised to recombinant proteins and by antibodies made to full-length gp120 purified from virus. The antigen employed was a synthetic peptide containing 15 amino acids, representing amino acid residues 308-322, RIQRGPGRAFVTIGK, of env gp120 (HTLV-IIIB isolate). Five of the monoclonal antibodies raised to this antigen have reactivity with gp120 from divergent strains of HIV-1 in Western blot assays. The two of these five which were tested with live cells infected with the divergent HIV-1 isolates IIIB, MN, and RF were specifically reactive by fluorescence analyses with cells infected with the MN and IIIB isolates. Four of the five monoclonal antibodies blocked the fusion of IIIB-infected cells with uninfected MOLT-4 target cells. The monoclonal antibody most reactive with MN-infected cells by fluorescence, #5025A, blocked the fusion of MN-infected cells with uninfected MOLT-4 cells. Four of the five monoclonal antibodies neutralized the IIIB isolate of HIV-1 in vitro, but none neutralized the MN or RF isolates at the levels of antibody tested (less than or equal to 50 micrograms/ml). Taken together these data indicate that monoclonal antibodies to the immunodominant neutralizing domain of HIV-1 gp120 display different levels of group reactivity depending on the assay system being examined.

We have studied the biologic and physical properties of a monoclonal antibody that binds to gp120, the exterior envelope glycoprotein of the human immunodeficiency virus (HIV) strain HTLV-IIIB. Designated 9284, the antibody possesses viral neutralizing activity and inhibits syncytium formation by infected cells. The antibody recognized a region of the polypeptide backbone previously described as an important neutralizing epitope. This region lies 307-330 residues from amino terminus of the glycoprotein. We have compared the biologic and physical properties of this antibody to those of the recently described 0.5 beta monoclonal antibody to gp120. The 0.5 beta antibody was biologically more potent and bound an epitope slightly downstream to that of the 9284 antibody. The antibodies did not differ significantly in their affinity for gp120. In competition studies, the 0.5 beta antibody was displaced by the 9284 antibody, but the binding of the latter was unaffected by 0.5 beta.

Tetanus toxoid (TT) reactive CD4+ cells were infected with HTLV-IIIB and exposed to TT at various times throughout a 7-day interval. Acute infection per se failed to produce overt cytopathology. However, exposure of infected cells to TT resulted in a rapid loss of cell viability, an increase in viral p24 expression, and a decline in T-cell blastogenesis. To determine whether HIV infection of antigen presenting cells (APC) could impact on T-cell activation, virus infected APC were utilized to present TT to responsive CD4+ cells. Use of infected APC produced effects similar to antigen stimulation of infected T-cells. These results suggest that the conditions of antigen presentation during T-cell activation may provide an excellent opportunity for virus transmission which may produce maximal immune dysfunction. However, preincubating antigen specific T-cells with the virostatic agent 3'-azido-3'-deoxythymidine (AZT) could prevent most of these effects.

Cytophilic antibodies which mediate antibody dependent cellular cytotoxicity (ADCC) against envelope antigens of human immunodeficiency virus (HIV) can be found in seropositive individuals. In these experiments, sera from a wide spectrum of HIV infected patients ranging from asymptomatic to overt acquired immunodeficiency syndrome (AIDS) were shown to contain high titers of antibodies that mediate ADCC. Not only did patient antibodies bind to surface expressed viral antigens and mediate ADCC against cells chronically infected with human T-lymphotropic virus type IIIB (HTLV-IIIB), but also against cells infected with the divergent HTLV-IIIRF2 and HTLV-IIIMN viral isolates. Similar results were obtained with target cells bearing purified GP 120 from HTLV-IIIB and HTLV-IIIRF2, indicating that a major portion of the activity was mediated by anti-GP 120 antibodies. Consistent with this was the ability to absorb most of the group-specific ADCC activity from the serum of an HIV infected individual using affinity columns bearing purified HTLV-IIIB GP 120. The finding that human antibodies reactive against the HIV envelope glycoprotein mediate ADCC against cells chronically infected with divergent strains of HIV will have important implications in designing rational approaches to passive and active immunotherapy.

In efforts to identify B cell and T cell epitopes of HIV-1 structural components, serum as well as lymphocytes from HIV-1-seropositive individuals were reacted with several recombinant and native peptides representing defined viral gag and env determinants. Several areas of discordance between humoral and cellular reactivity were identified. Specifically, the principal neutralizing site within HIV-1, the major envelope glycoprotein gp120, failed to elicit detectable cellular reactivities. The carboxyl portion of gp120 and the transmembrane gp41 region were uniformly recognized by patient antibodies but did not produce significant lymphocyte blastogenesis. However, the amino half of gp120 elicited cellular responses in a majority of the immunocompetent individuals tested, despite its extremely low reactivity with patient sera. Last, the major HIV-1 structure component p24 was found to be the most consistent T cell activation antigen among the panel tested.

A rapid, simple, reproducible and semi-quantitative assay to measure neutralizing antibodies has been developed. It employs a unique cell line which is exquisitively sensitive to infection with all HIV isolates tested. The assay is amenable to microtiter formulation as well as analysis by automation.