Cutting edge: CD4 is not required for the functional activity of IL-16.

IL-16 functions as a chemoattractant factor, inhibitor of HIV replication, and inducer of proinflammatory cytokine production. Previous studies have suggested that CD4 is the receptor for IL-16, because only CD4+ cells respond to IL-16 and both the anti-CD4 Ab OKT4 and soluble CD4 can block IL-16 function. However, these are only indirect evidence of a requirement for CD4, and to date a direct interaction between IL-16 and CD4 has not been shown. In this paper, we report that cells from CD4 knockout mice are as responsive to IL-16 as their CD4 wild-type equivalents in both assays testing for IL-16 function (chemotaxis and production of proinflammatory cytokines). In addition, the inhibitory effect of soluble CD4 on IL-16 function observed using CD4 wild type murine cells was not observed using CD4 knockout cells. These data demonstrate that CD4 is not required for IL-16 function and suggest that another molecule acts as the major receptor.

Decline of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocytes in the peripheral blood of long-term nonprogressing macaques infected with SIVmac32H-J5.

The evolution of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocyte precursors (CTLps) and their relationship with virus replication were studied in SIV-infected macaques. After primary viremia, 3 of 8 macaques lost culturable virus and polymerase chain reaction-detectable provirus in peripheral blood. Although proviral DNA persisted in the spleen and lymph nodes, virus loads were below or barely above detection levels. Throughout the study, the 3 macaques remained asymptomatic, with stable CD4+ cell counts. These findings were associated with the detection of CTLps directed against both structural and regulatory SIV proteins. The response peaked during the first 7 months of infection but waned subsequently. CTLps increased after rechallenge of 1 macaque, suggesting that limited antigenic stimulation contributed to their disappearance from circulation. Transient viremia with increasing CTLp frequencies and antibody titers also suggested at least partial susceptibility to reinfection. These findings bear implications for vaccination strategies aimed at inducing protective CTLs against lentiviruses.

Simian immunodeficiency virus (SIV) from sun-tailed monkeys (Cercopithecus solatus): evidence for host-dependent evolution of SIV within the C. lhoesti superspecies.

Recently we reported the characterization of simian immunodeficiency virus (SIVlhoest) from a central African l'hoest monkey (Cercopithecus lhoesti lhoesti) that revealed a distant relationship to SIV isolated from a mandrill (SIVmnd). The present report describes a novel SIV (SIVsun) isolated from a healthy, wild-caught sun-tailed monkey (Cercopithecus lhoesti solatus), another member of the l'hoest superspecies. SIVsun replicated in a variety of human T-cell lines and in peripheral blood mononuclear cells of macaques (Macaca spp.) and patas monkeys (Erythrocebus patas). A full-length infectious clone of SIVsun was derived, and genetic analysis revealed that SIVsun was most closely related to SIVlhoest, with an amino acid identity of 71% in Gag, 73% in Pol, and 67% in Env. This degree of similarity is reminiscent of that observed between SIVagm isolates from vervet, grivet, and tantalus species of African green monkeys. The close relationship between SIVsun and SIVlhoest, despite their geographically distinct habitats, is consistent with evolution from a common ancestor, providing further evidence for the ancient nature of the primate lentivirus family. In addition, this observation leads us to suggest that the SIVmnd lineage should be designated the SIVlhoest lineage.

Chemical inactivation of recombinant vaccinia viruses and the effects on antigenicity and immunogenicity of recombinant simian immunodeficiency virus envelope glycoproteins.

The efficiency of paraformaldehyde (PFA) and binary ethylenimine (BEI) in inactivating recombinant vaccinia virus (rVV), present in baby hamster kidney cells expressing simian immunodeficiency virus envelope glycoproteins (SIV-Env), was measured in a series of inactivation studies. Both compounds were shown to be effective in reducing rVV titres. The use of standard 3-day titration assays proved to be inadequate to measure PFA inactivation, since upon prolonged incubation, residual rVV infectivity was detected in cultures negative at 3 days. Different procedures using PFA or BEI were selected to assess their influence on the antigenicity and immunogenicity or rVV expressed SIV-Env. Antigenicity, as defined by the ability to react with a panel of monoclonal antibodies recognizing major antigenic sites, and immunogenicity, as defined by the ability to induce SIV envelope specific and virus neutralizing serum antibodies in rats, proved to be preserved after either inactivation procedure. These data show that both protocols using PFA or BEI can be used successfully as part of the procedures to remove residual rVV infectivity.

SIVagm infection of its natural African green monkey host.

Possible reasons for the apathogenicity of simian immunodeficiency virus (SIVagm) in its natural African green monkey (AGM) host were investigated. In most respects, the SIVagm/AGM system was shown to resemble human immunodeficiency virus type 1 (HIV-1) infection of humans. AGMs were shown to respond to infection with immune responses similar to those seen in HIV-1-infected humans, with no obvious controlling mechanism observed. The rate of SIVagm in vivo variability was likewise shown to be consistent with that described for HIV-1. Similarly, the level of infection in the peripheral blood was reminiscent of the level in asymptomatic HIV-1-infected patients, although never reaching the levels associated with AIDS. Some potentially important differences were, however, observed. Like humans, AGM CD8+ cells secrete a factor able to suppress SIVagm (and HIV-1) replication but unlike humans, AGMs have a very high percentage of CD8+ lymphocytes in circulation. Also, unlike humans during the asymptomatic stages of infection, AGM lymph nodes do not seem to act as a reservoir for SIVagm and the lymph node structure is not affected. Whether these phenomena are causative or incidental to the state of apathogenicity is the subject of further investigations.

Vaccine-induced virus-neutralizing antibodies and cytotoxic T cells do not protect macaques from experimental infection with simian immunodeficiency virus SIVmac32H (J5).

To gain further insight into the ability of subunit vaccines to protect monkeys from experimental infection with simian immunodeficiency virus (SIV), two groups of cynomolgus macaques were immunized with either recombinant SIVmac32H-derived envelope glycoproteins (Env) incorporated into immune-stimulating complexes (iscoms) (group A) or with these SIV Env iscoms in combination with p27gag iscoms and three Nef lipopeptides (group B). Four monkeys immunized with recombinant feline immunodeficiency virus Env iscoms served as controls (group C). Animals were immunized intramuscularly at weeks 0, 4, 10, and 16. Two weeks after the last immunization, monkeys were challenged intravenously with 50 monkey 50% infectious doses of virus derived from the J5 molecular clone of SIVmac32H propagated in monkey peripheral blood mononuclear cells. High titers of SIV-neutralizing antibodies were induced in the monkeys of groups A and B. In addition, p27gag-specific antibodies were detected in the monkeys of group B. Vaccine-induced cytotoxic-T-lymphocyte precursors against Env, Gag, and Nef were detected on the day of challenge in the monkeys of group B. Env-specific cytotoxic-T-lymphocyte precursors were detected in one monkey from group A. In spite of the observed antibody and T-cell responses, none of the monkeys was protected from experimental infection. In addition, longitudinal determination of cell-associated virus loads at weeks 2, 4, 6, 9, and 12 postchallenge revealed no significant differences between vaccinated and control monkeys. These findings illustrate the need to clarify the roles of the different arms of the immune system in conferring protection against primate lentivirus infections.

Antigenicity and immunogenicity of recombinant envelope glycoproteins of SIVmac32H with different in vivo passage histories.

Shortly after infection of two rhesus monkeys (Macaca mulatta) either with a SIVmac32H challenge stock or with the same virus that had been passaged in another rhesus monkey for 11 months, SIV-envelope genes were cloned from their peripheral blood mononuclear cells and subsequently expressed by recombinant vaccinia viruses. The molecular weights and antigenicities of the thus produced envelope glycoproteins were largely identical to those of the native SIV. The envelope glycoprotein derived from the in vivo passaged virus proved to be poorly recognized by virus neutralizing monoclonal antibodies directed against one of the seven antigenic sites for which monoclonal antibodies were available. Immunization studies in rats showed that this protein was also less efficient in inducing antibodies against this antigenic site, and that it induced significantly lower levels of virus neutralizing antibodies than the other SIV-envelope glycoprotein. The immunogenicity of the SIV-envelope glycoprotein incorporated into immune stimulating complexes (iscoms) was compared to that of the same protein presented with Quil A or MDP-tsl.

Rapid inactivation of HIV-1 in single donor preparations of human fresh frozen plasma by methylene blue/light treatment.

Human fresh frozen plasma (FFP) was spiked with highly titered HIV-1 and illuminated with visible light in the presence of 1 microM of the photoactive dye methylene blue (MB). As shown by titration on MT-4 cells, the infectivity of the virus containing plasma was rapidly lost during illumination: after 5 min the infective titer was reduced by 4.3 and after 10 min by at least 6.32 log10, i.e. it was below the detection limit of the assay applied. Methylene blue without illumination and illumination alone had only a marginal effect on HIV-1 infectivity. Thus our data indicate that the MB/light treatment of FFP is an effective method to eliminate the risk of HIV-1 infection through use of the product. This is especially important for those cases in which the plasma is collected during the 'window period' between infection of the donor and the subsequent seroconversion.

Insufficient inactivation of HIV-1 in human cryo poor plasma by beta-propiolactone: results from a highly accurate virus detection method.

Human cryo poor plasma was spiked with high titre HIV-1 and subjected to a beta-propiolactone (beta-PL) inactivation procedure. The residual titre of HIV-1 was measured using a sensitive and accurate titration technique based upon the killing of MT-4 cells by HIV and the subsequent inability to incorporate 3H-thymidine. It was found that under these conditions a reduction of only 1 log virus titre was achieved after one hour exposure to beta-PL and of 1.8 logs after 5 h. The presence of viable HIV was confirmed using immunohistochemical techniques, by transfer of live virus to fresh cells, and by production of reverse transcriptase. These results have obvious implications for products where human plasma is treated with beta-PL as the only virus inactivation step.

Quantitation of a lentivirus in its natural host: simian immunodeficiency virus in African green monkeys.

We have examined the viral load in the peripheral blood of simian immunodeficiency virus (SIV)-infected African green monkeys with a view to the unexplained apathogenicity of African green monkey SIV (SIVagm) in its natural host. By using polymerase chain reaction, viral DNA was detected in fresh peripheral blood mononuclear cells (PBMC) of each of nine seropositive animals. The virus DNA load was variable among the monkeys tested, ranging from 5 to 50 (mean = 15) copies per 10(5) PBMC, which is comparable to that of human immunodeficiency virus type 1 (HIV-1) in humans. The level of infectious SIVagm in PBMC was measured by endpoint dilution cultures. SIVagm was recovered from PBMC from 14 of 17 antibody-positive monkeys (82%), and the mean SIVagm titer in PBMC of seropositive African green monkeys was 10 tissue culture infectious doses per 10(6) cells, similar to the titer shown for HIV in asymptomatic carriers. Free infectious virus was isolated from the plasma of 4 of 17 monkeys (24%), and SIVagm expression in peripheral blood in vivo, as demonstrated by in situ hybridization, was detectable only in those animals which were viremic. SIVagm replication is therefore not totally suppressed in vivo, and SIVagm has a viral load equivalent to that seen for HIV-1 in asymptomatic humans.

AIDS vaccine: present problems and future perspectives.

Vaccination has proved to be an effective means for the prevention of infectious diseases. Advances in our understanding of the human immunodeficiency virus (HIV) and the immune system of the host may lay the foundation for the development of an AIDS vaccine. Current attempts to develop vaccines focus on the development of substances that will produce a different type of immune response from that which occurs naturally. Progress has been made in understanding the mechanisms by which the AIDS virus stimulates an neutralizing antibody response and triggers specific cytotoxic T lymphocytes in the host. The pressing need for a vaccine has prompted the testing of several candidate vaccines based on the simian immunodeficiency virus (closely related to HIV) in the macaque animal model for AIDS. The lessons learned from these trials will be valuable for developing future vaccines.

Vaccine protection against SIVmac infection by high- but not low-dose whole inactivated virus immunogen.

Eight rhesus macaques were immunized intramuscularly four times (0, 1, 2, and 4 months) over a period of 4 months with a formalin-inactivated whole Simian immunodeficiency virus (SIV) vaccine in the presence of muramyl dipeptide (MDP) as adjuvant. Four animals received 0.5 mg and the other four received 0.1 mg immunogen per injection. Three weeks after the final immunization, the vaccinated monkeys along with two control monkeys were challenged intravenously with 50 MID50 of SIVmac251-32H. At the time of challenge, one monkey from the high dose group had a high titer (1:761) of antibody able to neutralize in vitro the homologous 32H strain of SIV mac. All other animals had lower but measurable titers (1:57-1:453), although there was no correlation between the levels of neutralizing antibody and subsequent protection. Upon challenge, three of four animals from the low dose group (plus the nonvaccinated control animals) became infected as demonstrated by reisolation of virus from peripheral blood mononuclear cells, by SIVmac-specific polymerase chain reaction, and by the development of a strong anamnestic response. In the sera from these animals the titer of neutralizing antibody rose to over 1:5,100. All other animals (one from low dose group and all four of the high dose group) remained negative by all parameters at 4 months post challenge. These data indicate that when used in conjunction with MDP, the amount of immunogen required per immunization for protection against challenge is between 0.1 and 0.5 mg.

Mutational analysis of the simian immunodeficiency virus SIVmac nef gene.

We are using site-directed mutagenesis of single viral genes to identify and analyze the genetic determinants of human and simian immunodeficiency virus pathogenicity. In a first approach, we have constructed a series of simian immunodeficiency virus SIVmac nef mutants by partial deletion and insertions in the nef gene, as this gene is a candidate gene for the establishment and maintenance of latency. nef insertion mutants replicated faster than wild-type SIVmac, suggesting that the nef gene product acts as a negative factor for replication. Surface phenotyping revealed that cultures permanently infected with nef mutants exhibit an enhanced expression of viral proteins on the outer cell surface. We have analyzed the properties of the mutant viruses in cell culture and intend to use rapidly replicating mutants (putatively unable to undergo latency) as model vaccine viruses in the rhesus monkey.

Neutralizing antibodies and antigens in AIDS.

Until recently, much of the effort put into development of an AIDS vaccine has focussed on the elicitation of a neutralizing antibody response. The viral target of neutralization, HIV envelope glycoprotein, has been produced in bulk through recombinant techniques, but has had little success as a vaccine. The specific epitopes to which neutralizing antibodies bind have been mapped, and although the major epitope is hypervariable, others are conserved. This allows the design of second generation vaccines. Meanwhile, vaccine studies in the SIV animal model simply using inactivated virus as immunogen have demonstrated that an effective vaccine is at least possible. A variety of HIV vaccine preparations are now under investigation and the outlook for the future is promising.

Immunological studies of the basis for the apathogenicity of simian immunodeficiency virus from African green monkeys.

Potential reasons for the lack of pathogenicity of the simian immunodeficiency virus SIVagm in its natural host, the African green monkey (AGM, Cercopithecus aethiops), were investigated with respect to immunological mechanisms. The functional immune response of monkeys to infection was similar (though not identical) to that of humans to infection with human immunodeficiency virus type 1 (HIV-1). In the sera of infected animals, neutralizing antibodies were found to be low or absent, and in particular there was no neutralization of the various isolates by homologous sera. There was no detectable antibody/complement cytotoxicity, though AGM sera were able to initiate antibody-dependent cellular cytolysis of infected cells in the presence of healthy effector peripheral blood lymphocytes. As in the human/HIV system, macrophages from AGMs are readily infected by SIVagm. Two possibly important differences between the AGM/SIVagm system and the human/HIV system are (i) the low immune response of the AGMs to the core protein of SIVagm and (ii) the significantly lower inhibitory effect of SIVagm proteins on the proliferation of AGM lymphocytes.

Decreased accessory cell function of macrophages after infection with human immunodeficiency virus type 1 in vitro.

Peripheral blood monocytes from human immunodeficiency virus (HIV)-infected individuals or AIDS-related complex/AIDS patients ex vivo exhibit distinct alterations in some but not all immune functions. In studies presented here, monocytes from healthy donors were infected with HIV 1 in vitro and co-cultures with autologous uninfected T lymphocytes were set up. The monocyte/macrophage (M phi)-dependent T cell function was determined by measurement of proliferative and secretory [interleukin (IL)2, interferon-gamma] responses to lectin (phytohemagglutinin), mitogen (anti-CD3 monoclonal antibody), or recall antigen (tetanus toxoid, tuberculin). Accessory function of M phi was normal after HIV infection when optimal amounts (10%-20%) were added to the T lymphocytes. However, HIV infection of M phi significantly decreased T cell proliferative responses and secretion of IL2 when supplemented at limited dilution (0.5%-5%), although interferon-gamma production was not affected. Whereas the lipopolysaccharide-triggered M phi production of IL1 was not impaired by HIV 1 infection, there was a significant decrease in this response when anti-CD3 monoclonal antibody or tetanus toxoid were used to trigger the peripheral blood mononuclear cells. The impairment of proliferation of T lymphocytes in the presence of HIV 1-infected M phi could be overcome by addition of exogenous IL 1. Taken together, these data clearly show that the mononuclear phagocyte-dependent enhancement of stimulated T cell proliferation and lymphokine secretion is decreased when the restricted numbers of monocytes/M phi are HIV 1 infected. There are, therefore, two possible roles of M phi in HIV infection and progression to disease. First, as a reservoir and vehicle for dissemination of the virus, and second, as an immune cell whose essential functions are impaired by infection.

Demonstration of cross-reactive antibodies able to elicit lysis of both HIV-1- and HIV-2-infected cells.

A total of 100% of sera from a large number of HIV-1-infected patients contained antibodies able to elicit Antibody-dependent cellular cytotoxicity lysis of cells infected with the HIV-1 isolates IIIB or RF. Levels of activity could not be correlated with activities in ELISA or neutralizing antibody assays nor with the clinical status of the patients. Surprisingly, 8 of 156 patients sera could additionally elicit lysis of HIV-2-infected cells, and cold target competition assays demonstrated that the cross-reactivity was apparently mediated via recognition of common epitope(s) expressed on the surface of cells infected with either group of HIV. The ADCC mechanism was shown to be mediated by a CD16+ lymphocyte. This demonstration of an effector mechanism able to attack and eliminate cells infected with a wide range of HIV strains has obvious implications for development of putative vaccines.

Productive infection of both CD4+ and CD4- human cell lines with HIV-1, HIV-2 and SIVagm.

Human monolayer cells of various origins were shown to be susceptible to infection by HIV-1, HIV-2 and simian immunodeficiency virus obtained from African green monkeys (SIVagm). Immunoperoxidase staining revealed infection of 2-7% of the monolayer cells, although in order to achieve infection approximately 50-fold more virus was necessary than with CD4(+)-permissive lymphoma cells. No CD4-receptor antigen expression by fibroblastoid cells was detectable by immunofluorescence using several monoclonal antibodies (MAbs), although a low level of CD4-specific messenger RNA expression was revealed by Northern analysis (with the exception of Tera-1 and RD cells). Attempts to block viral infection by anti-CD4 MAbs indicated a CD4 receptor-mediated mechanism for all lines tested except RD cells. We conclude that a low level of CD4-receptor expression is sufficient to allow infection of fibroblastoid cells. The infectability of a CD4-negative cell line indicates a second pathway of cellular infection, possibly mediated by a cellular receptor distinct from the CD4 molecule.