Redefining the HIV-infectious window period in the chimpanzee model: evidence to suggest that viral nucleic acid testing can prevent blood-borne transmission.

BACKGROUND: Although it is rare, blood-transmitted HIV infection can occur when a donor presents in the window period between HIV-1 exposure and the first appearance of detectable p24 antigen. STUDY DESIGN AND METHODS: To study this seronegative window period, a chimpanzee (X034) was inoculated with 38 median tissue culture infective doses of HIV-1 IIIB; serum and peripheral blood mononuclear cells were obtained one to two times per week for 12 weeks and then biweekly for 12 weeks. Infectivity was monitored by the detection of serum HIV RNA, cell-associated HIV DNA, p24 antigen, and anti-HIV and by co-culture methods. RESULTS: No HIV markers were noted until 5 weeks after inoculation, at which time virus was isolated and HIV RNA and DNA were detected in plasma and cells, respectively. Anti-HIV and HIV p24 antigen were not present until 8 weeks after inoculation. Plasma and cells obtained from Chimpanzee X034 3 or 4 weeks after exposure were then sequentially inoculated into a second chimpanzee (X176); no HIV infection was observed in this animal during serial follow-up for 24 weeks after each inoculation. In contrast, when the fifth-week HIV-1 RNA- and DNA-positive sample was inoculated, Chimpanzee X176 was unequivocally infected with HIV-1. CONCLUSIONS: Nucleic acid testing narrowed the seronegative window by 3 weeks (37%). More important, there was no demonstrable infectivity in either plasma or peripheral blood mononuclear cells obtained before molecular markers were detectable. This suggests that the infectious window may be considerably shorter than the total window as measured from exposure and that nucleic acid testing might not only shorten the seronegative window, but totally prevent transfusion-transmitted HIV infection.

Induction of neutralizing antibodies to T-cell line-adapted and primary human immunodeficiency virus type 1 isolates with a prime-boost vaccine regimen in chimpanzees.

Five chimpanzees were immunized by administration of one or more intranasal priming doses of one to three recombinant adenoviruses containing a gp160 insert from human immunodeficiency virus type 1 (HIV-1) MN (HIV-1MN) followed by one or more boosts of recombinant HIV-1SF2 gp120 delivered intramuscularly with MF59 adjuvant. This regimen resulted in humoral immune responses in three of five animals. Humoral responses included immunochemically active anti-H1V-1 antibodies (Abs) directed to recombinant gp120 and neutralizing Abs reactive with T-cell-line-adapted HIV-1MN and HIV-1SF2. In addition, neutralizing activity was detected to the two homologous primary isolates and to two of three heterologous primary isolates which, like the immunizing strains, can use CXCR4 as a coreceptor for infection. The three animals with detectable neutralizing Abs and a fourth exhibiting the best cytotoxic T-lymphocyte response were protected from a low-dose intravenous challenge with a cell-free HIV-1SF2 primary isolate administered 4 weeks after the last boost. Animals were rested for 46 weeks and then rechallenged, without a boost, with an eightfold-higher challenge dose of HIV-1SF2. The three animals with persistent neutralizing Abs were again protected. These data show that a strong, long-lived protective Ab response can be induced with a prime-boost regimen in chimpanzees. The data suggest that in chimpanzees, the presence of neutralizing Abs correlates with protection for animals challenged intravenously with a high dose of a homologous strain of HIV-1, and they demonstrate for the first time the induction of neutralizing Abs to homologous and heterologous primary isolates.

CD8+ cell-mediated immune responses: relation to disease resistance and susceptibility in lentivirus-infected primates.

Immune responses mediated by CD8+ lymphocytes have been correlated with protection from HIV infection and disease progression in humans and nonhuman primates. The CD8+ cell population is heterogeneous in terms of biological function and phenotype. We have undertaken a review of the current state of knowledge of subtypes of CD8+ cells and their role in immune responses directed to HIV and related primate lentiviruses. Differences in the pathogenesis of lentivirus infections in various primate hosts were examined and the possible roles of the various subpopulations of CD8+ lymphocytes in the resistance and/or susceptibility to lentivirus-related disease were compared.

An immunovirological study of central nervous system involvement during HIV-1 infection of chimpanzees.

Chimpanzees infected with human immunodeficiency virus type 1 (HIV-1) are used to model acquired immunodeficiency syndrome (AIDS). Since the central nervous system (CNS) is involved in AIDS, we performed an immunovirological study in 18 chimpanzees inoculated up to 87 months prior to the study (mean, 45 months) with HIV-1 and 8 uninfected controls. Serum and cerebrospinal fluid (CSF) IgG and albumin levels of infected chimpanzees never exceeded those of controls. The CSF/serum albumin ratio was elevated in 1 of 18 infected chimpanzees compared to controls; however, all animals had an elevated ratio indicating a more open blood-brain barrier relative to humans. The intrathecal IgG production index was elevated in only 1 of 18 infected chimpanzees compared to controls. Identical serum and CSF IgG bands were found by isoelectric focusing in 2 of 8 controls and in 1 of 18 infected chimpanzees. None of these bands reacted with recombinant HIV-1 p24gag or gp 120env. HIV-1 was isolated from the peripheral blood of 4 of 18 infected chimpanzees but never from the paired CSF samples. Anti-HIV-1 antibody was detected by a enzyme-linked immunosorbent assay in 18 of 18 paired serum and CSF samples and by Western blot in 18 of 18 serum and 13 of 18 CSF samples from infected chimpanzees without a difference in pattern. Polymerase chain reaction analysis on brain tissue of one animal was negative for HIV-1 sequences. Our results demonstrate that, unlike human infection, chimpanzees inoculated with HIV-1 show no evidence of isolatable virus in the CSF and no evidence of intrathecal anti-HIV-1 antibody synthesis up to several years after experimental infection. The lack of CNS involvement may contribute to the delay or suppression of clinical disease in infected chimpanzees.

Immunogenicity of recombinant influenza virus haemagglutinin carrying peptides from the envelope protein of human immunodeficiency virus type 1.

Haemagglutinin (HA), the major surface glycoprotein of influenza virus, is a potent immunogen against which viral neutralizing antibodies are directed. Studies of the three-dimensional structure of HA have identified major antigenic sites on the molecule. We have exploited HA as a carrier for small antigenic regions (epitopes) of the HIV-1 envelope (env) glycoprotein. Using recombinant DNA techniques, the epitopes were inserted in-frame into a known antigenic site of HA to produce HA-epitope chimeras. Guinea-pigs and mice immunized with these chimeras in combination with adjuvant generated significant immune responses against the carrier HA and also produced epitope-specific antibodies that recognized the native whole HIV-1 env. One of the chimeras which contained a V3-loop sequence of HIV-1 env elicited neutralizing antibodies against the homologous strain of HIV-1. The antibodies against HA and the inserted epitopes remained at high levels for up to 72 weeks. Remarkably, these responses were generated with low doses of immunogens containing only nanogram quantities of the inserted epitopes. These results suggest the utility of HA as a carrier to allow selective antibody induction against foreign epitopes, and offer a new approach for vaccine development as well as for the production of monospecific antibodies useful in diagnostics and research.

Construction and characterization of adenovirus co-expressing hepatitis B virus surface antigen and interleukin-6.

Coexpression of biologically active interleukin 6 (IL-6), an immunoregulator, and hepatitis B virus surface antigen (HBsAg), an immunogen, was obtained using an adenovirus type 7 (Ad7) vector. Two recombinant adenoviruses (re-Ad) containing both the HBsAg and IL6 genes were constructed: one virus was capable of expressing IL6 with its signal peptide (spIL6) (Ad7::spIL6::HBsAg), and the second virus lacked this sequence (Ad7::IL6::HBsAg). A third recombinant contained only HBsAg (Ad7::HBsAg). All three Ad constructs were plaque purified and characterized in the A549 human lung cell line. The growth kinetics of the recombinants were similar to wild-type (wt) Ad7. The production and secretion of HBsAg (p24 and gp27) from cells infected with each re-Ad were at a level greater than 9 micrograms/10(6) cells by 118 h postinfection. Two IL-6 of approx. 24 and 27 kDa were produced and secreted into the culture medium from cells infected with Ad7::spIL6::HBsAg, and maximal accumulation occurred by 92 h p.i. at a level > 260 ng/10(6) cells. One cell-associated IL-6 of approx. 23 kDa was produced from cells infected with Ad7::IL6::HBsAg at a level > 12 ng/10(6) cells. Importantly, the Ad-produced IL-6 were determined to be biologically active by enhancing immunoglobulin production in lymphoblastoid cells. The co-production of IL-6 with HBsAg did not affect growth of these recombinant Ad, immunoreactivity of HBsAg, or the biological activity of IL-6 in tissue culture cells.

Chimpanzees in AIDS research: a biomedical and bioethical perspective.

The present article represents a consensus view of the appropriate utilization of chimpanzees in AIDS research arrived at as a result of a meeting of a group of scientists involved in AIDS research with chimpanzees and bioethicists. The paper considers which types of studies are scientifically justifiable in this species, the conditions under which such studies should be carried out, and the conditions which should be encouraged for post-experimental retirement of these animals.

Chimpanzees in AIDS research: a biomedical and bioethical perspective.

The present article represents a consensus view of the appropriate utilization of chimpanzees in AIDS research arrived at as a result of a meeting of a group of scientists involved in AIDS research with chimpanzees and bioethicists. The paper considers which types of studies are scientifically justifiable in this species, the conditions under which such studies should be carried out, and the conditions which should be encouraged for post-experimental retirement of these animals.

Sodium appetite in response to sodium deficiency in baboons.

Naive male baboons exhibit little hedonic intake of 300 mM NaCl but develop a robust Na appetite with Na deficiency. With the first episode of Na deficiency, increased drinking of saline solution occurs in the first 1-3 days, but correction of deficit is slow over 5-6 days. Na intake and repair of deficit become more rapid with experience. After three episodes of Na deficiency, the baboons immediately drink Na solution when given access and repair the deficit over 1-2 days. These experimental data showing an innate Na appetite in primates are important in relation to behavior of gorillas and chimpanzees in the wild and anthropological records of behavior toward salt sources. They open an avenue for physiological analysis of cerebral mechanisms subserving Na appetite in primates and humans.

Social factors influencing performance of a foraging task for captive chimpanzees.

The performance of captive chimpanzees (Pan troglodytes) during a simulated foraging activity was compared to that reported for the foraging behavior of wild chimpanzees. The ability to find hidden fruit in a large outdoor play area was measured for 34 subjects housed in ten separate groups. Sex differences were apparent, with females searching for and finding significantly more fruit than males did. Chimpanzees of high and medium dominance rank found more fruit than those of low rank. Neither age nor stage of the female sexual cycle exerted an influence. The subjects became more proficient at finding fruit during the second block of trials. The results reflect possible influences of captivity on chimpanzee social behavior.

HIV-1 expression in chimpanzees can be activated by CD8+ cell depletion or CMV infection.

CD8+ cell antiviral activity and cytomegalovirus (CMV) were investigated in vivo as possible cofactors influencing the outcome of HIV-1 infection. The role of CD8+ cell suppression of HIV replication was evaluated by depleting CD8+ cells in two infected chimpanzees by inoculation with monoclonal anti-CD8 antibodies. Two other infected animals were injected with chimpanzee CMV (CCMV)-infected human fibroblasts to determine if exposure to this virus would induce HIV replication. Treatment with anti-CD8 antibody resulted in recovery of virus from the CD4+ lymphocytes of one animal at 1, 4, and 6 months, and from a second animal at 1 month postinoculation. In contrast, virus had been recovered only once or not at all from these infected chimpanzees for 4 years prior to treatment. Similarly, HIV was recovered from the CD4+ cells of the two animals 2 to 3 months after inoculation of CCMV-infected fibroblasts but not after inoculation of control uninfected fibroblasts. These studies suggest that CD8+ cell-mediated suppression and the presence of other viruses (such as CMV) could act as cofactors in influencing the extent of HIV-1 replication in vivo and, possibly, progression to disease.

Coexpression of the simian immunodeficiency virus Env and Rev proteins by a recombinant human adenovirus host range mutant.

Recombinant human adenoviruses (Ads) that replicate in the intestinal tract offer a novel, yet practical, means of immunoprophylaxis against a wide variety of viral and bacterial pathogens. For some infectious agents such as human immunodeficiency virus (HIV), the potential for residual infectious material in vaccine preparations must be eliminated. Therefore, recombinant human Ads that express noninfectious HIV or other microbial proteins are attractive vaccine candidates. To test such an approach for HIV, we chose an experimental model of AIDS based on simian immunodeficiency virus (SIV) infection of macaques. Our data demonstrate that the SIV Env gene products are expressed in cultured cells after infection with a recombinant Ad containing both SIV env and rev genes. An E3 deletion vector derived from a mutant of human Ad serotype 5 that efficiently replicates in both human and monkey cells was used to bypass the usual host range restriction of Ad infection. In addition, we show that the SIV rev gene is properly spliced from a single SIV subgenomic DNA fragment and that the Rev protein is expressed in recombinant Ad-SIV-infected human as well as monkey cells. The expression of SIV gene products in suitable live Ad vectors provides an excellent system for studying the regulation of SIV gene expression in cultured cells and evaluating the immunogenicity and protective efficacy of SIV proteins in macaques.

Selection of the chimpanzee over the baboon as a model for Helicobacter pylori infection.

Baboons (Papio sp.) and chimpanzees (Pan troglodytes) were screened for the presence of Helicobacter pylori. The gastric mucosae of the baboons were colonized by large spiral bacteria. However, a group of adult chimpanzees were identified that were free of spiral gastric bacteria, with five animals being recruited into an H. pylori challenge study. These animals were inoculated orogastrically with one of four strains of H. pylori and followed for up to 26 weeks. H. pylori was established in one of these animals during a primary challenge and in two other animals on secondary challenge. It was shown that the chimpanzee can be infected with H. pylori and that the inflammatory response in these animals mimics that seen in humans. Infection was marked by an antibody response to H. pylori-specific antigens in two animals. It was observed that H. pylori antibody-negative chimpanzees had no apparent infection by H. pylori or related bacteria. Thus serological screening of chimpanzees can be used to identify candidate animals for further evaluation.

Antibodies are produced to the variable regions of the external envelope glycoprotein of human immunodeficiency virus type 1 in chimpanzees infected with the virus and baboons immunized with a candidate recombinant vaccine.

Chimpanzees infected with human immunodeficiency virus type 1 produce antibodies against the variable regions of the external envelope glycoprotein gp120. All five variable regions contain an epitope which is recognized by at least one of five chimpanzee sera. Each of the sera recognized a different pattern of epitopes. It is suggested that this varying response contributes to the emergence of variant viruses in the host. In contrast with the variability of the chimpanzees' response to replicating virus, that of baboons to a candidate recombinant vaccine is more uniform. Baboons injected with recombinant gp120 produced high levels of antibodies to epitopes within both the variable and conserved regions which coincided with epitopes previously shown to induce neutralizing antibodies.

Nasal capsule shape changes following septopremaxillary ligament resection in a chimpanzee animal model.

Recent human data suggest a relationship between the disruption of the septopremaxillary ligament (SPL) attachment and lack of anterior midfacial growth in cleft lip and palate (CLP) individuals. Early SPL resection in chimpanzees resulted in premaxillary growth deficits through 1200 days. Since the SPL is also continuous with the nasal bones, the present study was undertaken to investigate compensatory nasal capsule shape changes following SPL resection in a chimpanzee animal model. The study used 17 chimpanzees (Pan troglodytes): seven unoperated controls, five sham surgical controls, and five animals with early (average 144 days) SPL resection. Lateral head x-ray films and dental study models were collected quarterly and landmarks representing boundaries of the nasal capsule were identified. Mean nasal capsule polyhedrons were constructed from linear measurements at 200, 600, and 1000 days. In animals with SPL resection, the nasal capsule appeared truncated, shortened anteroposteriorly, and nasion was displaced posteriorly compared to controls by 600 days. Tensor biometric analysis of the growth/shape changes of the nasal capsule triangles revealed no significant differences across age in SPL resection animals while significant (p less than .05) age-related changes were noted in both control groups. Results showed that early SPL resection resulted in maintaining the neonatal nasal capsule morphology in the chimpanzee and suggested that such early growth mechanisms may be operating in complete CLP individuals as well. These data support the concept of early re-establishment of the SPL in primary nasolabial cleft repair to facilitate midfacial and nasal capsule growth.

CD4 immunoadhesins in anti-HIV therapy: new developments.

CD4, the cell-surface receptor for the human immunodeficiency virus (HIV), is a member of the immunoglobulin (Ig) gene superfamily. It contains 4 extracellular sequences homologous to Ig variable domains, the first of which (V1) is sufficient for binding to HIV. To develop CD4 as an anti-HIV therapeutic, we engineered a CD4 immunoadhesin (CD4-IgG)--a fusion protein containing the V1 and V2 domains of CD4 with the hinge and Fc regions of human Ig heavy chain. A chimeric protein of this type has several advantages compared to the soluble receptor, including a greatly extended in vivo half-life and greater avidity for HIV; moreover, like an antibody, it performs effector functions via its Fc domains, such as complement activation and antibody-dependent cell-mediated cytotoxicity. In vivo experiments show that CD4-IgG protects against HIV-I IIIB infection of chimpanzees when administered prior to viral challenge. In addition, CD4-IgG is transferred efficiently across the placenta from mother to fetus in rhesus monkeys. To evaluate its safety in humans, we conducted a phase-I clinical trial in adult patients with AIDS and AIDS-related complex. We found that, in a total of 16 patients, administration of CD4-IgG was well tolerated at doses up to 1000 micrograms/kg of body weight, with no important clinical or immunological toxicities noted. Given its unique properties, particularly the ability of CD4-IgG to cross the placenta, we plan to focus future clinical efforts on preventing infection of newborns via maternal-fetal transfer of HIV.

A comparative study of natural killer cell activity, lymphoproliferation, and cell phenotypes in nonhuman primates.

Three different species of nonhuman primates (baboons [Papio hamadryas], rhesus monkeys [Macaca mulatta], and African green monkeys [Cercopithecus aethiops]) were evaluated for their natural killer cell activity, and for the ability of their peripheral blood mononuclear cells to proliferate in response to known mitogens (concanavalin A, phytohemagglutinin, and pokeweed mitogen) and to react with a panel of mouse monoclonal antibodies directed against human leukocyte surface antigens. Rhesus monkeys displayed the highest natural killer cell cytotoxic activity (185.7 +/- 33 lytic units) compared with those of baboons (83.8 +/- 19 lytic units) and of African green monkeys from West Africa (39.08 +/- 8 lytic units) and from the Caribbean basin (37.9 +/- 9 lytic units). No correlation was observed between the natural killer cell cytotoxic activity and the percentage of CD16+ natural killer cells among the three species studied. High spontaneous proliferative capacity was observed in African green monkeys obtained from West Africa compared with those of the other species studied. Although no significant differences were noted in T and B cell mitogen-induced in vitro proliferation, baboon mononuclear cells were less responsive to concanavalin A (stimulation index of 16 +/- 3 [mean +/- standard error of mean]) than to phytohemagglutinin (stimulation index of 47 +/- 12). However, rhesus and African green monkey cells proliferated more efficiently in response to concanavalin A. Unlike in human beings where the ratio between helper-inducer (CD4+) and cytotoxic-suppressor (CD8+) T-lymphocytes is generally greater than 1, the CD4+/CD8+ ratios in baboons and rhesus and African green monkeys were 0.58, 0.69, and 0.35, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Native but not denatured recombinant human immunodeficiency virus type 1 gp120 generates broad-spectrum neutralizing antibodies in baboons.

The protection of individuals from human immunodeficiency virus type 1 (HIV-1) infection with an envelope subunit derived from a single isolate will require the presentation of conserved epitopes in gp120. The objective of the studies presented here was to test whether a native recombinant gp120 (rgp120) immunogen would elicit responses to conserved neutralization epitopes that are not present in a denatured recombinant gp120 antigen from the same virus isolate. In a large study of 51 baboons, we have generated heterologous neutralizing activity with native, glycosylated rgp120SF2 but not with denatured, nonglycosylated env 2-3SF2. After repeated exposure to rgp120SF2 formulated with one of several adjuvants, virus isolates from the United States, the Caribbean, and Africa were neutralized. The timing of the immunization regimen and the choice of adjuvant affected the virus neutralization titers both quantitatively and qualitatively. These results suggest that vaccination with native, glycosylated rgp120 from a single virus isolate, HIV-SF2, may elicit a protective immune response effective against geographically and sequentially distinct HIV-1 isolates.