Potentiation of simian immunodeficiency virus (SIV)-specific CD4(+) and CD8(+) T cell responses by a DNA-SIV and NYVAC-SIV prime/boost regimen.

T cell-mediated immune responses play an important role in the containment of HIV-1 replication. Therefore, an effective vaccine against HIV-1 should be able to elicit high frequencies of virus-specific CD8(+) and CD4(+) T cells. The highly attenuated poxvirus-based vaccine candidate, NYVAC-SIV-gag-pol-env (NYVAC-SIV-gpe), has been shown to induce and/or expand SIV-specific CD4(+) and CD8(+) T cell responses in both naive and infected macaques. In this study, the immunogenicity of NYVAC-SIV-gpe alone was compared with a combination regimen where priming with an optimized DNA-SIV-gag-env vaccine candidate was followed by a NYVAC-SIV-gpe boost. In macaques immunized with the prime-boost regimen, the extent and durability of CD8(+) T cell response to an immunodominant SIV gag epitope was increased and these animals recognized a broader array of subdominant SIV epitopes in the cytolytic assay. In addition, the prime-boost regimen significantly enhanced the proliferative responses to both SIV gag and env proteins. Thus, the combination of these vaccine modalities may represent a valuable strategy in the development of a vaccine for HIV.

Persistent infection of rhesus macaques by the rev-independent Nef(-) simian immunodeficiency virus SIVmac239: replication kinetics and genomic stability.

We generated previously a Nef(-), replication-competent clone of SIVmac239 in which the Rev protein and the Rev-responsive element were replaced by the constitutive transport element (CTE) of simian retrovirus type 1 (A. S. von Gegerfelt and B. K. Felber, Virology 232:291-299, 1997). In the present report, we show that this virus was able to infect and replicate in rhesus macaques. The Rev-independent Nef(-) simian immunodeficiency virus induced a persistent humoral immune response in all monkeys, although viral loads were very low. Upon propagation in the monkeys, the genotype remained stable and the virus retained its in vitro growth characteristics. The infected monkeys showed normal hematological values and no signs of disease at more than 18 months post-virus exposure. Therefore, replacement of the essential Rev regulation by the CTE generated a virus variant that retained its replicative capacity both in vitro and in vivo, albeit at low levels.

Replacement of posttranscriptional regulation in SIVmac239 generated a Rev-independent infectious virus able to propagate in rhesus peripheral blood mononuclear cells.

Lentiviruses control virion production via posttranscriptional regulation mediated by the viral Rev protein. In this study, we demonstrate that the Rev regulation of SIVmac239 can be replaced by the presence of the cis-acting transport element (CTE) of the type D simian retroviruses 1 (SRV-1). To avoid the possibility of generating revertants, the Rev-Independent SIV clones have both rev and the Rev responsive element (RRE) destroyed by multiple point mutations that do not affect the overlapping tat and env open reading frames. Virus stocks generated from these Rev-independent SIV molecular clones can infect and can be propagated in rhesus peripheral blood mononuclear cells (PBMCs). Therefore, the Rev/RRE system of SIVmac239 can be replaced by the SRV-1 CTE as previously shown for HIV-1. In both rhesus and human primary cells, the replicative capacity of the Rev-independent SIV is 10- to 20-fold lower than that of the wild-type virus. Rhesus PBMC-derived virus stocks of the Rev-independent SIV have lower infectivity. Interestingly, in CEM x 174 cells, no difference in replicative capacity between wild-type and Rev-independent SIV has been observed. The Rev-independent SIV has a stable genotype after several passages in primary cells. The availability of such Rev-Independent viruses will allow the study of the role of Rev in pathogenesis and the potential generation of attenuated SIV strains.

Identification of cross-reactive antigenic target regions for HIV type 1-specific antibody-dependent cellular cytotoxicity.

Monkey-derived hyperimmune antisera against 40 peptides, together representing the entire envelope gp120 of HIV-1LAI, were used to map antibody-dependent cellular cytotoxicity (ADCC) target regions. Four regions corresponding to amino acids 65-126, 152-230, 248-330, and 445-466 were found to contain epitopes inducing ADCC activity not only against HIV-1LAI-infected cells but also against cells infected with HIV-1SF2 and clinical isolates of HIV-1. When comparing seroreactivity to the individual peptides with ADCC titers none of the regions seemed to be dominant. These results thus describe cross-reactive regions involved in the functional immunity against HIV-1 gp120.

Broadly reactive HIV-2 and SIVmac specific antibody-dependent cellular cytotoxicity in immunized and infected cynomolgus monkeys.

Antibody-dependent cellular cytotoxicity (ADCC) was analysed in groups of cynomolgus monkeys that had been immunized with either HIV-2 (strains SBL6669 or SBL-K135) or SIVmac. HIV-2- and SIVmac-infected monkeys were also studied for ADCC. Sequential serum samples were collected from the animals, which were followed for 1 to 3 years. Sera from the HIV-2-immunized monkeys had ADCC against both homologous and heterologous HIV-2 strains as well as cross-reactivity against SIVmac-infected target cells. This broadly reactive ADCC response could be detected within the first weeks after immunization. Homologous ADCC was also seen in seven of eight SIVmac-immunized monkeys which were all protected from later challenge with SIVmac or SVsm. ADCC titres sometimes decreased after a few months if the immunized monkeys were not boosted whereas most of the HIV-2-and SIVmac-infected monkeys developed a rapid and persistent ADCC response. The presence of ADCC, like the presence of neutralization in previous studies, did not predict whether the immunized monkeys would be protected upon challenge with live virus.

Specificity of antibody-dependent cellular cytotoxicity in sera from human immunodeficiency virus type 2-infected individuals.

ADCC activity in sera from HIV-2-infected individuals was monitored against HIV-1IIIB, SIVmac, and three different HIV-2 strains. The sera mediated ADCC against the HIV-2 strains in high frequencies and reacted equally well with SIVmac, whereas no cross-reactivity was seen against HIV-1IIIB. The degree of antigenic similarities between the virus strains was also evaluated in order to estimate the variability of ADCC target regions. The SIVmac strain and two of the HIV-2 strains were antigenically more similar to each other whereas another HIV-2 strain appeared more distantly related with regard to ADCC target regions. Although strain-specific ADCC was present in some HIV-2-positive sera, HIV-2 ADCC was more broadly reactive and appeared in higher frequencies against these specific strains than has been previously shown for HIV-1 ADCC in a group of four HIV-1 strains. The difference was, however, not significant. To further delineate target regions for ADCC the sera were tested against peptides representing different regions of the HIV-2 envelope protein. The V3 region and the C-terminal end of gp125 were thus suggested to be involved in ADCC. Target regions for HIV-2-specific ADCC may only partly overlap with HIV-2-neutralizing regions since the two activities were not always present in the same sera. Characterization of broadly reacting immune responses like HIV-2-specific ADCC and identification of their specific target epitopes is essential for the development of an efficient AIDS vaccine.

Autologous neutralizing antibodies prevail in HIV-2 but not in HIV-1 infection.

Human immunodeficiency virus type 2 (HIV-2) has been reported to be less pathogenic than HIV-1. We have investigated the capacity of sera from nine HIV-2-infected individuals to neutralize their own autologous virus. All nine HIV-2-infected individuals neutralized autologous virus with titers ranging between 20 and 320. In contrast, we have previously reported that most HIV-1-infected individuals lack such antibodies. The difference between HIV-1 and HIV-2 infection was statistically significant (P < 0.0002, Pearson test) and the difference in neutralizing antibody prevalence may explain the faster disease progression in HIV-1-infected individuals than in HIV-2-infected individuals.

Lack of autologous neutralizing antibodies in the cerebrospinal fluid of HIV-1 infected individuals.

The cerebrospinal fluids (CSF) and sera from HIV-1-infected individuals at different clinical stages were monitored for neutralizing activity against CSF-derived HIV-1 isolates. None of the CSF samples and only one of seven serum samples could neutralize the autologous CSF isolate. CSF samples collected one to two years later from the same patients also lacked autologous neutralizing antibodies against these isolates. However, some CSF samples were able to neutralize heterologous CSF isolates albeit in low titers. HIV antibody positive control sera could readily neutralize all of the CSF isolates demonstrating that these isolates were not resistant to neutralization per se. IgG antibodies against the HIV-1 envelope protein and, specifically, against the V3 loop of HIV-1 gp120 (MN) were present in some CSF samples, although the samples lacked neutralizing activity. In summary, this study demonstrates a lack of autologous neutralizing antibodies in CSF samples when assayed against CSF-derived HIV-1 isolates.

Identification of human neutralization-inducing regions of the human immunodeficiency virus type 1 envelope glycoproteins.

Four major neutralizing regions of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein were identified and characterized with a panel of 80 HIV-1 antibody-positive human sera. Levels of neutralizing antibodies against the HIV-1 strains IIIB, SF2, and RF were compared with reactivity in ELISAs against peptides that correspond to certain regions of the HIV-1 envelope. A correlation between high neutralizing activity and strong seroreactivity against specific peptides suggested that the corresponding regions might be involved in neutralization. This was further substantiated by using peptides to inhibit neutralization by a panel of 10 HIV-1 antibody-positive sera. The positions of three neutralizing sites, defined earlier mostly by antisera from animals, were confirmed in the present study. Human sera thus recognize the strain-specific third variable region of gp120 (amino acids 304-318), the C-terminal end of gp120 (amino acids 489-508), and the conserved region in the intracellular part of gp41 (amino acids 732-746). It is likely that these different regions mediate help rather than self-sufficient neutralization. Furthermore, a human neutralizing region was detected in a conserved part of gp41 (amino acids 647-671). Accordingly, neutralizing antibodies directed to this region were found to be cross-reactive between HIV-1 strains. Peptides corresponding to these four regions were able to inhibit neutralization mediated by serum from HIV-1 antibody-positive individuals. These results indicate that this conserved B-cell epitope of the HIV-1 envelope elicits a virus-neutralizing antibody response during natural infection in humans and may therefore be considered for inclusion in a vaccine against HIV-1.

Analysis of the V3 loop in neutralization-resistant human immunodeficiency virus type 1 variants by direct solid-phase DNA sequencing.

To study the molecular basis for the emergence of human immunodeficiency virus type 1 (HIV-1) variants with reduced sensitivity to neutralization by autologous sera, the DNA sequence of the envelope V3 loop was determined in HIV-1 isolates derived from four patients with primary HIV-1 infection and sequentially thereafter. The gene fragment encoding the V3 loop of gp120 was amplified by a nested polymerase chain reaction (PCR) and subsequently sequenced by a novel solid phase DNA sequencing approach allowing direct sequencing of the viral genome without the need for previous cloning. The results show that all patients have HIV-1 with unique primary sequence of the V3 loop and antibodies to this structure are produced at seroconversion. The structural analysis also demonstrates that the mechanism for virus escape from neutralization in vivo is complex. Thus, in one patient the emergence of neutralization-resistant viruses coincided with the introduction of several amino acid changes in the V3 loop, while in two other patients the V3 loop remained completely unchanged. These findings suggest that an understanding of the interaction between the humoral immune response and HIV-1 may require detailed structural studies of the entire envelope.

Isolate-specific neutralizing antibodies in patients with progressive HIV-1-related disease.

Four individuals with increasing severity of HIV-1 infection were studied for serum neutralizing activity against their own virus isolates collected during 1-3 years. Sequential serum samples and HIV-1 isolates were available from these patients from the stage of lymphadenopathy to severe immunodeficiency. The replicative capacity of isolates changed from slow/low to rapid/high in each patient during the study period. Sequential virus isolates showed differences in sensitivity to neutralization by autologous as well heterologous area. Taken together with our previous results demonstrating that variant viruses resistant to neutralization by autologous sera emerge during the year following primary infection, neutralization-resistant variants seem to emerge during the entire course of HIV-1 infection. The ability to produce neutralizing antibodies to autologous virus appears to correlate with replicative capacity of the virus and the degree of immunodeficiency in the patient.

Biological characterization of infectious molecular clones derived from a human immunodeficiency virus type-1 isolate with rapid/high replicative capacity.

In order to molecularly characterize rapidly and slowly replicating HIV-1 variants, molecular clones were obtained from a rapid/high virus isolate. This isolate, 4803, had only been passaged in peripheral blood mononuclear cells (PBMC) prior to cloning. Molecular cloning was done in bacteriophage lambda-dash using high molecular weight DNA of isolate 4803 infected PBMC. Seven recombinant phages were identified. The clones were found to be related to each other and differed only at 1 or 2 restriction sites (out of 28). The molecular clones were transfected into various cell types by electroporation. The phenotype of progeny viruses was found to be dependent on the cell type used for transfection. Progeny viruses produced by PBMC cultures differed from the parental isolate in that they did not form syncytia and lacked the capacity to replicate in cell lines. Since transfection of PBMC yielded progeny viruses within 1 week, this phenotype is considered to be the true phenotype of the clones. Transfection of the T-lymphoid HUT-78 cell line and of the monocytoid U937-2 cell line yielded progeny viruses after considerable delay (more than 1 month). Progeny viruses from HUT-78 cells were similar to the parental isolate in that they formed syncytia in PBMC and replicated in all cell lines tested. Progeny viruses from U937-2 cells showed an intermediate phenotype in that they replicated in U937-2 but not in T-lymphoid cell lines. These results indicate that molecular clones of a rapid/high virus may have a restricted replicative capacity compared to the parental, genetically heterogenous virus isolate.

In vitro maturation of mononuclear phagocytes and susceptibility to HIV-1 infection.

In the present report, we have studied the in vitro transition of normal blood monocytes to macrophages by changes in cell morphology, and the expression of surface antigens with a panel of monoclonal antibodies. The maturation process was accompanied by notable changes in cell-surface markers in a time-dependent manner. The percentage of cells expressing CD11c, ICAM-1, HLA-DR, and Fc receptor class III increased while the CD4 and CD35 expression was markedly decreased. After demonstrating that in vitro monocytes mature to macrophages in a recognizable manner, we studied the susceptibility to HIV-1 infection at time points representing different stages of cell maturation. The results show that monocyte/macrophages are susceptible to HIV-1 infection at all stages of differentiation. However, the kinetics of virus replication depends on the degree of maturation at the time of infection. Two major patterns of replication were observed: Infection of monocytes resulted in efficient virus production measurable by reverse transcriptase activity in culture supernatant, whereas infection of fully differentiated macrophages yielded low but sustained virus release only demonstrable by p24 antigen assay. We were not able to detect differences in the capacity of the virus to infect and replicate in monocyte/macrophages with respect to cellular origin of the virus isolate and whether the viruses were laboratory-adapted strains or low-passaged patient isolates.

Naturally occurring HIV-1 isolates with differences in replicative capacity are distinguished by in situ hybridization of infected cells.

Replication of human immunodeficiency virus type 1 (HIV-1) isolates in peripheral blood mononuclear cells (PBMC) has been studied by in situ hybridization using the riboprobe BH10-R3 from HTLV-IIIB. Two series of isolates were tested: (a) 20 isolates from individuals with varying severity of HIV-1 infection and (b) sequential isolates from 5 subjects showing signs of clinical progression over a 45 month observation period. The results show that HIV-1 isolates with distinct replicative capacity can be distinguished by the intensity of radioactive labeling over single infected cells after in situ hybridization. Sequential isolates from patients with clinically progressive HIV-1 infection show a gradual increase in replicative capacity over time. In PBMC cultures infected with such sequential isolates, intensity of radioactive label over single infected cells increases and is strongest with isolates obtained at the time of low CD4 counts in blood. The results suggest that the restriction of virus replication that operates in the early stages of HIV-1 infection is gradually lost with progression of the disease.

Distinct replicative and cytopathic characteristics of human immunodeficiency virus isolates.

According to their capacity to replicate in vitro, human immunodeficiency virus (HIV) isolates can be divided into two major groups, rapid/high and slow/low. Rapid/high viruses can easily be transmitted to a variety of cell lines of T-lymphoid (CEM, H9, and Jurkat) and monocytoid (U937) origin. In contrast, slow/low viruses replicate transiently, if at all, in these cell lines. Except for a few isolates, the great majority of slow/low viruses replicate in peripheral blood mononuclear cells and Jurkat-tatIII cells constitutively expressing the tatIII gene of HIV-1. The viruses able to replicate efficiently cause syncytium formation and are regularly isolated from immunodeficient patients. Poorly replicating HIV isolates, often obtained from individuals with no or mild disease, show syncytium formation and single-cell killing simultaneously or, with some isolates, cell killing only.