Immunological reactivities of Ghanaian sera with HIV-1, HIV-2, and simian immunodeficiency virus SIVagm

To learn more about the relative prevalence of viruses from the human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) groups in Ghana, serum samples were collected in 1986 from 47 men and women with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC), 57 apparently healthy individuals, and 11 AIDS-free hospital inpatients. Western blot analysis revealed a total of 46 reactive sera. 43 of the 47 serum samples from those with AIDS or ARC were positive; 6 were seropositive for HIV-1, 18 for HIV-2, 17 for both HIV-1 and HIV-2, and the remaining 2 were not reactive with glycoproteins. Of the 2/10 individuals with mild symptoms of HIV infection who proved to be seropositive, 1 was positive for HIV-2 and 1 for HIV-1 and HIV-2. There were no seropositive reactions among the AIDS-free hospital patients, and only 1 such reaction (seropositive for HIV-2) among the healthy individuals. These findings indicate that both HIV-1 (6 cases in this series) and HIV-2 (20 cases) are responsible for the development of AIDS in Ghana, and that there is a high prevalence (18 cases) of cross-reactivity between the 2 viruses. There was no evidence of SIV infection. Further research is needed to determine whether these findings are a result of cross-reactivity between envelope proteins of HIV-1 and HIV-2, double infection of AIDS patients, or infection with a new variant strain. Since prostitutes comprised 25 of the 47 AIDS/ARC patients and 6 of the 10 with mild symptoms of HIV infection, they are an important target for preventive efforts.     

Isolation of simian virus 40 from rhesus monkeys (Macaca mulatta) with spontaneous progressive multifocal leukoencephalopathy.

Isolates of virus from the brain tissue of two naturally occurring cases of progressive multifocal leukoencephalopathy in rhesus monkeys (Macaca mulatta) have been characterized. Both isolates were demonstrated to be simian virus 40 (SV40) by serological tests and analysis of cleavage fragments of viral deoxyribonucleic acid produced by restriction endonuclease from Haemophilus influenzae. SV40 virions and the nonvirion T antigen were demonstrated in the brain lesions of one monkey by the fluorescent antibody staining technique. SV40 was not demonstrated in the brain of normal rhesus monkeys from the same colony with use of the same methods of viral isolation or demonstration of antigen.     

The principal neutralization determinant of simian immunodeficiency virus differs from that of human immunodeficiency virus type 1.

To identify the principal neutralization determinant (PND) of simian immunodeficiency virus (SIV), antisera were generated using recombinant gp110 [the SIV analog of the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein, gp120], gp140, several large recombinant and proteolytic envelope fragments, and synthetic peptides of the SIVmac251 isolate. When purified under conditions that retain its native structure, gp110 bound CD4 and elicited antisera that neutralized SIVmac251 with high titer. Native gp110 also completely inhibited neutralizing antibody in sera from SIVmac251-infected macaques. In contrast, denatured gp110 and gp140, large envelope fragments, and synthetic peptides (including peptides analogous to the HIV-1 PND) elicited very low or undetectable neutralizing antibody titers and did not inhibit neutralizing antibody in infected macaque sera. Enzymatically deglycosylated gp110 efficiently absorbed neutralizing antibodies from macaque sera, showing that neutralizing antibodies primarily bind the protein backbone. A 45-kDa protease digest product, mapping to the carboxyl-terminal third of gp110, also completely absorbed neutralizing antibodies from infected macaque sera. These results show that the PND(s) of this SIV isolate depends on the native conformation and that linear peptides corresponding to the V3 loop of SIV envelope, in contrast to that of HIV-1, do not elicit neutralizing antibody. This may affect the usefulness of SIVmac for evaluating HIV-1 envelope vaccine approaches that rely on eliciting neutralizing antibody.     

Expression of human CD4 in transgenic mice does not confer sensitivity to human immunodeficiency virus infection.

Transfection of the human CD4 molecule into mouse cells does not confer susceptibility to human immunodeficiency virus type 1 (HIV-1) infection. Expression of the human CD4 molecule in transgenic mice was seen to offer some new possibilities. However, transgenic mouse T cells expressing either the human CD4 receptor, or a hybrid human/mouse CD4 receptor alone or in conjunction with human major histocompatibility complex class I molecules, were refractory to in vitro HIV-1 infection. In addition, no infection was observed after in vivo HIV inoculation to mice of these various transgenic lines. Injection of recombinant gp160 viral protein to the transgenic mice did not alter their T and B cell populations. The existence of a dominant block in mouse cells that prevents HIV entry is discussed.     

Augmentation of immune responses to HIV-1 and simian immunodeficiency virus DNA vaccines by IL-2/Ig plasmid administration in rhesus monkeys

The potential utility of plasmid DNA as an HIV-1 vaccination modality currently is an area of active investigation. However, recent studies have raised doubts as to whether plasmid DNA alone will elicit immune responses of sufficient magnitude to protect against pathogenic AIDS virus challenges. We therefore investigated whether DNA vaccine-elicited immune responses in rhesus monkeys could be augmented by using either an IL-2/Ig fusion protein or a plasmid expressing IL-2/Ig. Sixteen monkeys, divided into four experimental groups, were immunized with (i) sham plasmid, (ii) HIV-1 Env 89.6P and simian immunodeficiency virus mac239 Gag DNA vaccines alone, (iii) these DNA vaccines and IL-2/Ig protein, or (iv) these DNA vaccines and IL-2/Ig plasmid. The administration of both IL-2/Ig protein and IL-2/Ig plasmid induced a significant and sustained in vivo activation of peripheral T cells in the vaccinated monkeys. The monkeys that received IL-2/Ig plasmid generated 30-fold higher Env-specific antibody titers and 5-fold higher Gag-specific, tetramer-positive CD8+ T cell levels than the monkeys receiving the DNA vaccines alone. IL-2/Ig protein also augmented the vaccine-elicited immune responses, but less effectively than IL-2/Ig plasmid. Augmentation of the immune responses by IL-2/Ig was evident after the primary immunization and increased with subsequent boost immunizations. These results demonstrate that the administration of IL-2/Ig plasmid can substantially augment vaccine-elicited humoral and cellular immune responses in higher primates.     

Factors contributing to the lack of human immunodeficiency virus type 1 (HIV-1) transmission in HIV-1-discordant partners

Correlates of resistance to infection by human immunodeficiency virus type 1 (HIV-1) are important for defining potential therapeutic interventions and for prophylactic vaccination. In this study, 11 couples discordant in their HIV-1 infection status were prospectively evaluated for the presence of protective factors. Behavioral characteristics of all subjects entailed a high risk of transmission. Cytotoxic T lymphocyte (CTL) responses against viruses isolated from the infected partner, and against laboratory virus isolates, were detected in 5 (45%) of 11 HIV-negative partners, including a CCR5Delta32-homozygous and a heterozygous subject. No CTL responses were observed in 6 control unexposed subjects. Marked variation in lymphocyte susceptibility to viral infection was noted. Resistance attributable to major histocompatibility complex discordance or anti-major histocompatibility complex antibodies was not identified. These results suggest that a combination of factors, including cellular immunity, viral characteristics, and coreceptor integrity, may be involved in the persistent nontransmission of HIV.     

Isolation of human immunodeficiency virus type 1 (HIV-1) from seropositive hemophiliac Japanese

We reported some biological properties of HIV-1 isolated from 16 hemophiliac Japanese and accidentally infected one mother. Peripheral mononuclear cells (PMCs) were obtained from them, one with AIDS, one with lymphadenopathy and the others were asymptomatic carriers. CD 8 depleted PMCs were obtained by panning methods. They were cocultivated with PHA-stimulated PMCs from seronegative donors. Fifteen HIV-1 isolates were obtained from 17 cases. Recovery rate was 87.5%. The replication rate of HIV-1 from AIDS patient was faster than other isolates from asymptomatic carriers. They did not from plaques on MT 4 cells. The host range study showed that all fifteen isolates infected primary macrophages and only two simultaneously infected human T cell line (MT 2). None of them showed infectivity to other T cell, B cell or monocytic cell lines. Although our study population was rather small, these results suggested that the majority of seropositive hemophiliac Japanese were already infected by HIV-1 and had the risk for the development of AIDS. Moreover, we recognized that HIV-1 from hemophiliac Japanese showed characteristic biological features, i,e, such as 1) weak cytopathic effects, 2) narrow host range and 3) tropism to primary macrophages. It is suggested that they may belong to a unique subtype of HIV-1 and their selective infectivity to primary macrophages have some relation to the clinical status of seropositive hemophiliacs. Further study is necessary to clarify these points.     

Antibodies to human immunodeficiency virus type 1 in the urine specimens of HIV-1-seropositive individuals

Specific antibodies to human immunodeficiency virus type 1 (HIV-1) were detected in 200-fold concentrated urine samples, but none were detected in unconcentrated urine specimens, from 100 randomly selected HIV-1--seropositive individuals by enzyme-linked immunosorbent assay (ELISA) and Western blot techniques using the manufacturer's recommended procedures. Using modified methods for both the ELISA and Western blot tests, antibodies to HIV-1 have also been detected in the unconcentrated urine specimens from the same HIV-1--seropositive individuals. No difference in the frequency of antibodies to HIV-1 were found between unconcentrated and 200-fold concentrated urine samples when tested by the modified methods. HIV-1 core antigen (p24) was not detected in either the concentrated or the unconcentrated HIV-1--seropositive adult urine samples; none of these individuals showed overt clinical or laboratory evidence of renal dysfunction. The titer of the antibodies to HIV-1 found in the urine specimens was found to be parallel with the titer of antibodies to HIV-1 in the corresponding individual's serum. Further elucidation of the pathophysiology and the nature of the specific antibodies to HIV-1 observed in the urine of HIV-1--seropositive individuals is under investigation in our laboratories.     

Production and of monoclonal antibodies to simian immunodeficiency virus envelope glycoproteins.

Eighteen monoclonal antibodies (MAb) to simian immunodeficiency virus (SIV) envelope have been characterized. All MAb were shown to bind to viral antigens on the surface of unfixed SIV-infected cells and to precipitate surface glycoproteins of SIVmac251. In Western blot 11 MAb bound to gp160 and gp120, five bound to gp160 and the transmembrane protein gp41 and two MAb did not react with denatured antigen. Preliminary competition assays identified the existence of six competition groups; two groups were within gp41 and four were within gp120. Of the latter four groups, three contained MAb with neutralizing activity. Two of the neutralizing MAb (KK5 and KK9) did not react with denatured antigen in Western blot suggesting that they may recognize conformational epitopes. Enzyme-linked immunosorbent-assay titres of MAb against SIVmac251 ranged from 10(2.4) to 10(5.6) and although similar titres were obtained with some MAb against other SIV and HIV antigens, the presence of isolate specific and shared group epitopes was demonstrated.     

Comparative evaluation of simian, simian-human, and human immunodeficiency virus infections in the pigtail macaque (Macaca nemestrina) model

The global impact of HIV/AIDS intensifies the need for a preventive vaccine and nonhuman primate models can help provide critical insights into effective immunity. Pigtail macaques (Macaca nemestrina) are increasingly studied as a nonhuman primate model for AIDS. We compared the virologic and immunologic characteristics of HIV-1, SIV, and SHIV infection of naive pigtail macaques across a series of preclinical HIV vaccine studies. SIVmac251 and SIVmac239 infection of naive pigtail macaques resulted in a gradual decline in peripheral CD4+ T cells in the setting of high levels of viremia, approximating most closely human infection of HIV-1. In contrast, the CXCR4-utilizing SHIVmn229 virus resulted in rapid depletion of CD4+ T cells and minimal generation of humoral or cellular immune responses, similar to that observed with SHIV89.6P infection of rhesus macaques. Infection with the CCR5-utilizing, rhesus macaque passaged, SHIVSF162P3 resulted in some overall CD4+ T cell decline, however, three of eight macaques naturally control SHIVSF162P3 viremia to very low levels in the setting of robust adaptive immunity. Despite attempts at infecting pigtail macaques with HIV-1 strains passaged in juvenile pigtail macaques in vivo or in PBMC isolated from pigtail macaques in vitro, only lower nonsustained levels of viral replication were observed. Our results provide a series of virologic models with which to evaluate potential AIDS vaccines in pigtail macaques.     

Copper inhibits the protease from human immunodeficiency virus 1 by both cysteine-dependent and cysteine-independent mechanisms.

The protease of the human immunodeficiency virus is essential for replication of the virus, and the enzyme is therefore an attractive target for antiviral action. We have found that the viral protease is inhibited by approximately stoichiometric concentrations of copper or mercury ions. Inactivation by Cu2+ was rapid and not reversed by subsequent exposure to EDTA or dithiothreitol. Direct inhibition by Cu2+ required the presence of cysteine residue(s) in the protease. Thus, a synthetic protease lacking cysteine residues was not inhibited by exposure to copper. However, addition of dithiothreitol as an exogenous thiol rendered even the synthetic protease susceptible to inactivation by copper. Oxygen was not required for inactivation of either the wild-type or the synthetic protease. These results provide the basis for the design of novel types of protease inhibitors.     

Lectin-carbohydrate interactions and infectivity of human immunodeficiency virus type 1 (HIV-1).

The aim of this study was to determine whether mannosyl-specific lectins, especially Concanavalin A (ConA), may bridge HIV-1 env glycoproteins to cell membranes to increase virus binding to its targets, and to what extent this lectin-carbohydrate interaction can modify HIV-1 infectivity for monocytic compared with lymphoid cells. Monocytic U937 and lymphoid CEM cells, which both express surface mannose, were utilized. Whether first incubated with env glycoprotein or with the cells, lectins bound both to the cells and to radiolabeled recombinant gp160 (rgp160). Thus, they enhanced rgp160 adsorption to the cells in a methyl-alpha-mannose inhibitable manner. ConA did not appear to bind to the V1 domain of CD4 at the U937 cell surface since Leu3a binding was not blocked in the presence of ConA, nor was recombinant CD4 retained on a ConA-agarose affinity matrix. Moreover, enhanced rgp160 binding to the cells was CD4 independent, since it was not modified by preincubating the cells with Leu3a. Finally, ConA did not inhibit the binding of CD4-IgG3 chimeric molecules to virions immobilized on nitrocellulose membrane, which argues against the possibility that it interferes with the interaction of gp120 and CD4. However, both when incubated with the virus or with the cells and despite mediating enhanced binding of env glycoprotein, ConA neutralized HIV-1 infectivity for monocytic U937 as well as for lymphoid CEM cells. In this respect, ConA behaves like neutralizing antibodies which do not interfere with CD4 binding of gp120 but rather with some later event that leads to virus entry. These findings obtained with plant lectins may be of relevance in vivo, inasmuch as endogenous mannosyl-binding proteins, which are known to function as opsonins, have been reported to inhibit in vitro infection by HIV-1.     

Kinetics of human immunodeficiency virus type 1 (HIV-1) DNA and RNA synthesis during primary HIV-1 infection.

HIV-1 replication and viral burden in peripheral blood mononuclear cells (PBMC) have been reported to be high in primary infection but generally very low during the prolonged period of clinical latency. It is uncertain precisely when this transition occurs during the HIV-1 infection and what the relationship is between the changes in HIV-1 replication versus the clearance of infected cells in the overall control of viral replication. In the present study, the kinetics of viral burden (i.e., frequency of HIV-1-infected cells) and replication during primary and early-chronic infection were analyzed in PBMC of four acutely infected individuals. High frequencies of HIV-1-infected cells and high levels of virus replication were observed in PBMC after primary HIV-1 infection. Down-regulation of virus replication in PBMC was observed in all four patients coincident with the emergence of HIV-1-specific immune responses. Other parameters of virus replication, such as circulating plasma p24 antigen and plasma viremia showed similar kinetics. In contrast, a significant decline in viral burden in PBMC was observed in only one of four patients. These results indicate that the down-regulation in the levels of virus replication associated with the clinical transition from acute to chronic infection does not necessarily reflect a reduction in viral burden, thus suggesting the involvement of additional factors. Identification of these factors will be important in elucidating the host mechanisms involved in the early control of HIV-1 infection and disease.