Effect of highly active antiretroviral therapy and thymic transplantation on immunoreconstitution in HIV infection.

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.

Monoclonal antibodies that bind to the core of fusion-active glycoprotein 41.

The heptad repeat regions HR1 and HR2 of HIV-1 gp41 can associate to form heterooligomers through helical coil-coil interactions that are believed to play a key role in virus-induced membrane fusion. The HR1/HR2 complex was proposed to be the core structure of the fusion-active conformation of gp41. Here, we show that two human monoclonal antibodies, Fab-d and 50-69, specifically recognize the putative fusion-active conformation of gp41. Fab-d binding requires the interaction between the HR1 and HR2 regions of gp41. The reactivity of human monoclonal antibody 50-69 to the C terminus of the HR1 sequence is dependent on the helical structure of HR1. It appears that HR2 is able to interact with HR1 and, subsequently, induce an epitope in HR1 that is required for 50-69 binding. Mutations that disrupt the helical structure of HR1 significantly compromise Fab-d and 50-69 binding. Although the epitopes are not identical, the ability of Fab-d to partially compete with 50-69 binding suggests a close proximity of the two epitopes. Antibodies that are able to interact with the core of the putative fusion-active gp41 may be useful in further unveiling the mechanism of HIV-induced membrane fusion.

Lymphokine-activated killer (LAK) cell anti-HIV-1 ADCC reactivity: a potential strategy for reduction of virus-infected cellular reservoirs.

Lymphocytes from HIV-1-seropositive and -seronegative individuals were examined to determine whether HIV-1 infection interfered with the ability to generate a lymphokine-activated killer (LAK) cell response. Following a 3-day ex vivo incubation in the presence of 1000 U/ml of recombinant interleukin-2, lymphocytes from seropositive individuals exhibited a LAK cell response which was equivalent to or greater than that of seronegative controls as measured against Daudi cell targets. LAK cells from seropositive and seronegative donors showed no specific cytolytic activity against gp120-coated or HIV-1-infected targets. However, in the presence of patient sera, significant levels of virus-specific LAK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) were observed. The level of this specific LAK cell-mediated ADCC was greater than that mediated under similar conditions by freshly isolated peripheral blood mononuclear cells. The greatest improvement in ADCC over baseline activity was seen with lymphocytes from AIDS patients after the 3-day ex vivo activation, suggesting that this patient population might benefit the most from adaptive LAK cell therapy.

Neutralizing antibodies in sera from macaques immunized with attenuated simian immunodeficiency virus.

Infection with attenuated simian immunodeficiency virus (SIV) in rhesus macaques has been shown to raise antibodies capable of neutralizing an animal challenge stock of primary SIVmac251 in CEMx174 cells that correlate with resistance to infection after experimental challenge with this virulent virus (M. S. Wyand, K. H. Manson, M. Garcia-Moll, D. C. Montefiori, and R. C. Desrosiers, J. Virol. 70:3724-3733, 1996). Here we show that these neutralizing antibodies are not detected in human and rhesus peripheral blood mononuclear cells (PBMC). In addition, neutralization of primary SIVmac251 in human and rhesus PBMC was rarely detected with plasma samples from a similar group of animals that had been infected either with SIVmac239Deltanef for 1.5 years or with SIVmac239Delta3 for 3.2 years, although low-level neutralization was detected in CEMx174 cells. Potent neutralization was detected in CEMx174 cells when the latter plasma samples were assessed with laboratory-adapted SIVmac251. In contrast to primary SIVmac251, laboratory-adapted SIVmac251 did not replicate in human and rhesus PBMC despite its ability to utilize CCR5, Bonzo/STRL33, and BOB/gpr15 as coreceptors for virus entry. These results illustrate the importance of virus passage history and the choice of indicator cells for making assessments of neutralizing antibodies to lentiviruses such as SIV. They also demonstrate that primary SIVmac251 is less sensitive to neutralization in human and rhesus PBMC than it is in established cell lines. Results obtained in PBMC did not support a role for neutralizing antibodies as a mechanism of protection in animals immunized with attenuated SIV and challenged with primary SIVmac251.

Neutralizing antibodies in sera from macaques infected with chimeric simian-human immunodeficiency virus containing the envelope glycoproteins of either a laboratory-adapted variant or a primary isolate of human immunodeficiency virus type 1.

The magnitude and breadth of neutralizing antibodies raised in response to infection with chimeric simian-human immunodeficiency virus (SHIV) in rhesus macaques were evaluated. Infection with either SHIV-HXB2, SHIV-89.6, or SHIV-89.6PD raised high-titer neutralizing antibodies to the homologous SHIV (SHIV-89.6P in the case of SHIV-89.6PD-infected animals) and significant titers of neutralizing antibodies to human immunodeficiency virus type 1 (HIV-1) strains MN and SF-2. With few exceptions, however, titers of neutralizing antibodies to heterologous SHIV were low or undetectable. The antibodies occasionally neutralized heterologous primary isolates of HIV-1; these antibodies required >40 weeks of infection to reach detectable levels. Notable was the potent neutralization of the HIV-1 89.6 primary isolate by serum samples from SHIV-89.6-infected macaques. These results demonstrate that SHIV-HXB2, SHIV-89.6, and SHIV-89.6P possess highly divergent, strain-specific neutralization epitopes. The results also provide insights into the requirements for raising neutralizing antibodies to primary isolates of HIV-1.

Evidence that antibody-mediated neutralization of human immunodeficiency virus type 1 by sera from infected individuals is independent of coreceptor usage.

Human immunodeficiency virus type 1 (HIV-1) uses a variety of chemokine receptors as coreceptors for virus entry, and the ability of the virus to be neutralized by antibody may depend on which coreceptors are used. In particular, laboratory-adapted variants of the virus that use CXCR4 as a coreceptor are highly sensitive to neutralization by sera from HIV-1-infected individuals, whereas primary isolates that use CCR5 instead of, or in addition to, CXCR4 are neutralized poorly. To determine whether this dichotomy in neutralization sensitivity could be explained by differential coreceptor usage, virus neutralization by serum samples from HIV-1-infected individuals was assessed in MT-2 cells, which express CXCR4 but not CCR5, and in mitogen-stimulated human peripheral blood mononuclear cells (PBMC), where multiple coreceptors including CXCR4 and CCR5 are available for use. Our results showed that three of four primary isolates with a syncytium-inducing (SI) phenotype and that use CXCR4 and CCR5 were neutralized poorly in both MT-2 cells and PBMC. The fourth isolate, designated 89.6, was more sensitive to neutralization in MT-2 cells than in PBMC. We showed that the neutralization of 89.6 in PBMC was not improved when CCR5 was blocked by having RANTES, MIP-1alpha, and MIP-1beta in the culture medium, indicating that CCR5 usage was not responsible for the decreased sensitivity to neutralization in PBMC. Consistent with this finding, a laboratory-adapted strain of virus (IIIB) was significantly more sensitive to neutralization in CCR5-deficient PBMC (homozygous delta32-CCR5 allele) than were two of two SI primary isolates tested. The results indicate that the ability of HIV-1 to be neutralized by sera from infected individuals depends on factors other than coreceptor usage.

Neutralizing antibody responses to human immunodeficiency virus type 1 in primary infection and long-term-nonprogressive infection.

The role of neutralizing antibodies in human immunodeficiency virus type 1 (HIV-1) infection is poorly understood and was assessed by evaluating responses at different stages of infection. Undiluted sera from long-term nonprogressors (LTNP) had broad neutralizing antibodies against heterologous primary isolates and were more likely to neutralize the contemporaneous autologous isolate than were sera from short-term nonprogressors and progressors. In primary infection, envelope-specific IgG was detected before the initial decline in plasma viremia, but neutralizing antibodies developed more slowly. Here, neutralizing antibodies against strains SF-2 and MN were sometimes the first to be detected, but titers were low for at least 17 weeks from onset of symptoms. Neutralizing antibodies against the early autologous isolate were detected for 4 patients by 5-40 weeks but were undetectable in 2 additional patients for 27-45 weeks. The results indicate that neutralizing antibody responses are slow to develop during primary infection and are uniquely broad in LTNP.

Clade B-based HIV-1 vaccines elicit cross-clade cytotoxic T lymphocyte reactivities in uninfected volunteers.

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.

IL-7 enhancement of antigen-driven activation/expansion of HIV-1-specific cytotoxic T lymphocyte precursors (CTLp).

CD8+ cytotoxic T lymphocytes are an important component in the immunologic control of human viral diseases. IL-7, a stromal cell-derived cytokine, has been demonstrated to enhance both anti-tumour and anti-viral CTL as well as lymphokine-activated killer (LAK) activity. We studied the ability of IL-7 to support the activation and the growth of in vitro antigen-specific CTL precursors (CTLp) present in peripheral blood mononuclear cells (PBMC) from HIV-infected patients. Results from these studies demonstrate that inclusion of IL-7 in a vaccinia/HIV-1 vector-based stimulation strategy greatly augmented overall CTL reactivities, whereas addition of IL-7 to unstimulated cultures failed to induce any significant anti-viral cytolytic activity. In four of six patients, HIV-specific lytic activities were significantly higher in cultures stimulated with antigen plus IL-7 compared with in vitro stimulation (IVS) with antigen alone. Cytotoxic activity was principally mediated by CD8+ effector cells, and CD3+/CD8+ cell expansion was increased by 2.7-fold in the presence of IL-7. In PBMC from seronegative donors, IL-7 enhanced anti-vaccinia CTL activities with less effect on cell proliferation. Furthermore, anti-gag CTL frequencies determined by limiting dilution analysis were increased by 2- and 10-fold in two asymptomatic patients following IVS plus IL-7 compared with antigen stimulation alone. Cytofluorimetric analysis revealed that IL-7 preferentially expanded CD8 memory cells (CD45RO+) and CD8+ lymphocytes expressing activation molecules. IL-7 was also able to support the growth of CD4+ lymphocytes, while having no effect on natural killer (NK)/K lymphocytes. Taken together, these data suggest that IL-7 acts cooperatively with the antigen supporting in vitro maturation of CTLp into functional cytotoxic effectors. Thus IL-7 may be an important biologic entity to consider as part of future immune-based therapies in which ex vivo expansion of antigen-driven CTL is an important determinant.

A molecular clasp in the human immunodeficiency virus (HIV) type 1 TM protein determines the anti-HIV activity of gp41 derivatives: implication for viral fusion.

We have previously reported that synthetic peptides representing the leucine zipper domain (DP107) and a second putative helical domain (DP178) of human immunodeficiency virus type 1 (HIV-1) gp41 exhibit potent anti-HIV activity. In this study we have used soluble recombinant forms of gp41 to provide evidence that the DP178 peptide and the DP178 region of gp41 associate with a distal site on the gp41 transmembrane protein whose interactive structure is influenced by the leucine zipper (DP107) motif. We also observed that a single coiled-coil-disrupting mutation in the leucine zipper domain transformed the recombinant gp41 protein from an inactive to an active inhibitor of HIV-1 fusion and infectivity, which may be related to that finding. We speculate that this transformation results from liberation of the potent DP178-related sequence from a molecular clasp with a leucine zipper, DP107, determinant. The results are discussed in the context of two distinct conformations for the gp41 molecule and possible involvement of these two domains in structural transitions associated with HIV-1-mediated fusion. The results are also interpreted to suggest that the anti-HIV activity of the various gp41 derivatives (peptides and recombinant proteins) may be due to their ability to form complexes with viral gp41 and interfere with its fusogenic processes.

Multiple CTL specificities against autologous HIV-1-infected BLCLs.

The cellular immune response to HIV-1 has been well studied but, in many respects, remains incompletely defined. Although CTL specificities against highly conserved HIV-1 determinants as dictated by vaccinia/HIV-1 vector constructs have been described, much less is known regarding patient cellular reactivities against autologous cells infected with HIV-1. One of the main obstacles in characterizing this cellular reactivity has been the absence of a targeting system which accurately represents the HIV infected cell in vivo and is, at the same time, adaptable for in vitro assays. Through the use of two separate strategies aimed at increasing cellular CD4 expression, we were able to infect B-lymphocyte cell lines (BLCLs) with multiple strains of HIV-1. HIV-1-infected BLCLs were recognized by autologous effector cells with cytolytic specificities against env, gag, or pol determinants. In addition, HIV-1-infected BLCLs were capable of eliciting in vitro CTL reactivities directed against env-, gag-, and pol-expressing targets. This cellular reactivity was mediated by CD8+ cells and was MHC Class I restricted, suggesting a classical CTL response. Since multiple antigens are recognized, an HIV-1-infected BLCL is a more natural representation of an in vivo cellular target than other available testing systems and should permit a more representative analysis of CTL responses during infection or following vaccination.

Studies of high doses of a human immunodeficiency virus type 1 recombinant glycoprotein 160 candidate vaccine in HIV type 1-seronegative humans. The AIDS Vaccine Clinical Trials Network.

We examined the safety and immunogenicity of a baculovirus-derived recombinant HIV-1 envelope glycoprotein vaccine candidate, rgp160 (VaxSyn; MicroGeneSys, Meriden, CT), administered at doses of 160 or 640 micrograms to 56 healthy, HIV-1-seronegative adults, in a randomized, double-blind, placebo-controlled study. Immunizations were given intramuscularly at 0, 1, 6, and 12 months. Both doses were generally well tolerated, although self-limited local reactions were frequent. No other clinical or laboratory toxicities were noted, and no effects on CD4 or CD8 lymphocyte counts or percentages were noted. Serum antibody responses to HIV proteins were detected by Western blot (WB) in 19 of 20 and in 19 of 19 recipients of four doses of 160 and 640 micrograms, respectively. Western blot responses developed more rapidly in the 640-micrograms group. High rates of EIA antibody responses to HIV-1 lysate were also present in both groups, and developed more rapidly in the 640-micrograms group. Enzyme immunoassay antibody responses to the immunogen (rgp160) were also frequent, but were infrequent to V3 to gp41 peptides. Neutralizing antibodies against the homologous HIV-1 LAI isolate were seen in 3 of 20 subjects (GMT = 11) who received four doses of 160 micrograms, and in 10 of 19 subjects who received four doses of 640 micrograms (GMT = 32). Fusion inhibiting antibody was not detected. CD4 blocking activity was seen in 3 of 19 subjects who received four doses of 640 micrograms. Complement-mediated antibody-dependent enhancement was found in sera from 11 of 19 volunteers in the 640-micrograms group. Lymphocyte proliferative responses to the immunogen were detected in 4 of 4 subjects tested, but no cytotoxic T cell activity was noted in 11 subjects. Administration of the 640-micrograms dose of this rgp160 vaccine candidate relative to the lower doses was associated with increased immunogenicity, including higher rates of homologous neutralizing antibody responses, although at low titer.

NIH conference. HIV vaccine development: a progress report.

The development of a safe, effective preventive vaccine for human immunodeficiency virus (HIV) infection remains an area of vigorous research. Several highly innovative vaccine candidates are being developed, and more than 13 vaccine candidates have been tested in human phase I or II trials. All have produced antibody and several have produced modest neutralizing titers, but to date no reproducible evidence has suggested prolonged, high-titer neutralization across a diversity of HIV strains. Furthermore, only the live recombinant vector approaches have produced some evidence of cytotoxic T-cell responses. The principal obstacle to progress is the lack of definitive information on what constitutes a protective immune response. There is no animal model for HIV-induced disease. Hence, the identification of the correlates of immunity and more useful animal models is among the highest priorities for HIV vaccine research. Large-scale efficacy trials raise daunting scientific, ethical, and resource issues. Nonetheless, preparation in such trials is underway in order to be in a position to evaluate the most promising vaccine candidate.