Jorg W. Eichberg

Suppression of human immunodeficiency virus replication by CD8+ cells from infected and uninfected chimpanzees

Over a 4-year period, infectious human immunodeficiency virus type 1 (HIV-1) has been recovered from cultured peripheral blood mononuclear cells (PBMC) of virus-infected animals only intermittently and at relatively low titers. In examining the possible mechanism for this observation, CD4+ cells or CD8+ cells were removed by panning from the PMBC before culture. A dramatic increase in frequency of HIV-1 recovery as well as in the level of virus replication was observed in the CD4+ cell-enriched or CD8+ cell-depleted cultures of PBMC from 3/3 infected animals. Moreover, addition of purified CD8+ effector cells from all 6 HIV-infected and 5/10 uninfected animals to an equal number of HIV-1 acutely infected purified CD4+ target cells resulted in 75-100% suppression of virus production. CD8+ cells from 3 additional uninfected animals caused delayed replication kinetics and moderate to low suppression of peak virus production. These findings contrast with the previously recognized absence of this HIV-1-suppressing activity of CD8+ cells from seronegative humans. This CD8+ cell-mediated suppression of viral replication could help explain the natural resistance of chimpanzees to HIV-induced disease.

Persistent infection of baboons and rhesus monkeys with different strains of HIV-2.

HIV-2 infection of eight rhesus macaques and two baboons was studied. Most animals were preselected for HIV-2 inoculation by testing their peripheral blood mononuclear cells (PBMC) for susceptibility to virus isolates from the Ivory Coast. The virus strains used (HIV-2UC2, HIV-2UC3, and HIV-2UC7) were also chosen by in vitro screening in PBMC for high replicating ability and cytopathicity. All the animals seroconverted within 2-4 weeks of infection and remained seropositive throughout the duration of the study. One macaque was sacrificed after 2 years, suffering from diarrhea and weight loss, and one baboon died of non-HIV-related causes. The remaining animals are asymptomatic, with normal CD4/CD8 ratios. Virus has been recovered from most animals, and persistent HIV-2 replication has been noted in three macaques and a baboon. Host range studies in T, B, and monocyte cell lines showed little or no differences between isolates obtained after inoculation and the original virus inoculum. However, isolates from the macaque that showed clinical symptoms were more cytopathic as reflected by plaque formation in MT-4 cells. The HIV-2-infected macaque or baboon could be useful as an animal model for elucidating the mechanisms of HIV pathogenesis and for evaluating potential antiviral therapies and vaccines.

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.

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.

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.

Lack of genetic restriction by a potential anti-idiotype vaccine for type B viral hepatitis

Anti-idiotype (anti-Id) reagents that bear an internal image capable of mimicking hepatitis B surface antigen (HBsAg) were used to induce an antibody to HBsAg (anti-HBs) response in both rabbits and chimpanzees. The anti-idiotype induced antibody response produced in rabbits recognized HBsAg determinants associated with the induction of protective immunity against hepatitis B virus (HBV). Attesting further to the specificity was the binding of the rabbit anti-idiotype to the anti-idiotype induced anti-HBs containing sera. Our findings suggest that genetic restrictions associated with the induction of an interspecies immune response may not be a limitation of anti-idiotype based vaccines. In addition, anti-idiotype immunization also produced an anti-HBs in chimpanzees, a species susceptible to infection with human HBV. These data demonstrate that internal-image-bearing anti-idiotype reagents can induce an immune response across species barriers. Additionally, the reagents represent a viable alternative approach to vaccination against agents such as hepatitis B virus that cause human disease.

In vivo transfer of cellular immunity to primates with transfer factor prepared from human or primate leucocytes.

Abstract Dialyzable transfer factor (TF d ) prepared from a human donor and transfer factor (TF) from baboon whole cell lysates was administered to 3 species of nonhuman primates: baboons, cebus monkeys and marmosets. In vivo transfer was evaluated with in vivo skin test and in vitro blastogenic responses to multiple antigens. Transfer of cellular reactivity in all three nonhuman primate species was demonstrated with both human TF d and baboon TF. A cumulative conversion rate of 45% for skin test responses and 65% for lymphocyte blastogenesis was demonstrated following human TF d injection while conversion was 17% and 33% respectively following baboon TF. Specificity was supported by the absence of conversion to TF donor negative antigens. There were no signficant differences observed between the 3 recipient primate species.

Cellular immunity in gnotobiotic primates induced by transfer factor.

Abstract Human dialyzable transfer factor (TF) was found capable of inducing in vivo (skin test) and in vitro blastogenesis) cellular immunity in gnotobiotic nonhuman primates. Because the animals were gnotobiotic (germ-free) and had not been skin tested previously, these data support the hypothesis that induction of cell-mediated immunity in recipients of TF does not require a “priming” exposure to specific and/or cross-reacting antigens, and that this induction may be due to an antigen-specific informational effect of TF. In addition, these results support the antigenic specificity of TF, in that recipient primates developed cellular reactivity only against donor “positive” but not against donor “negative” antigens.

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.

The influence of age and pregnancy on immune responses of baboons to mitogens and the baboon endogenous virus.

Abstract Immune responses to mitogens and baboon endogenous type C virus (BaEV) were assessed in baboons according to age, sex, and at various stages of pregnancy. Generally there were no significant differences noted in the immunologic responses between males and females. However, age and pregnancy had pronounced effects on mitogen responses and specific humoral and cell-mediated immunity (CMI) to BaEV. Lymphocyte responses to mitogens declined with increasing age and were significantly suppressed in the third trimester of pregnancy. Humoral immunity (HI) to BaEV assayed by radioimmunoprecipitation (RIP) was observed in pregnant, but not age-matched nonpregnant, baboons and this reactivity declined with increasing time of pregnancy. In nonpregnant animals, RIP antibodies were only detected in very young ( -12 years of age) baboons. CMI as assayed by lymphocyte blastogenesis (LB) and macrophage migration inhibitory factor (MIF) was suppressed during pregnancy. In relation to age LB responses to BaEV did not follow a clear pattern; however, MIF (like RIP) was only found in young (