Immune responses in baboons vaccinated with HIV-2 genetic expression libraries.

Immunization using genetic expression libraries may be an improvement over conventional DNA immunization using a single gene because more epitopes are simultaneously presented to the immune system. In this study, we evaluated the effectiveness of an HIV-2 vaccine made from a genomic expression library in baboons. We found that HIV-2 expression library immunization induced HIV-2-specific memory responses but low levels of CD8+ cell anti-viral responses and neutralizing antibodies. After intravenous virus challenge using a homologous pathogenic variant, HIV-2UC2/9429, viral loads were similar in the HIV-2-immunized and control baboons. We conclude that although immunization using HIV-2 expression libraries induces immune responses, this approach does not provide protection in baboons against intravenous challenge with HIV-2.

The nuclear factor kappa B and Spl binding sites do not appear to be involved in virus suppression by CD8 T lymphocytes.

CD8 T lymphocytes suppress primate lentivirus replication in a non-cytotoxic manner. This antiviral activity, mediated by a CD8 cell antiviral factor (CAF), involves an arrest in viral transcription. Present studies indicate that the CD8 T cell non-cytotoxic antiviral activity and CAF inhibit the replication of an SIV mutant virus lacking the nuclear factor kappa B (NF-kappaB) and Spl binding domains. The results strongly suggest that the NF-B and Spl binding sites are not involved in virus suppression by CD8 T lymphocytes.

Baboons as an animal model for human immunodeficiency virus pathogenesis and vaccine development.

Baboons (Papio cynocephalus) provide a valuable animal model for the study of human immunodeficiency virus (HIV) pathogenesis because HIV-2 infection of baboons causes a chronic viral disease that progresses over several years before clinical signs of acquired immunodeficiency syndrome (AIDS) appear. Since HIV-2-infected baboons develop a chronic viral infection, insights into the immuno-biology of viral latency, clinical stages of disease, virus infection of lymphatic tissue and HIV transmission can be gained using this animal model. The development of an AIDS-like disease in baboons is viral isolate and baboon subspecies dependent. Thus, viral virulence factors and host resistance can be studied as well as the mechanisms of innate and acquired immunity. The control of virus infection is dependent upon cytotoxic and non-cytotoxic antiviral activity of CD8+ T cells. In this regard, some of the HIV-2-infected baboons develop potent antiviral cellular immune responses that have a similar magnitude to that found in HIV-1-infected long-term survivors (or non-progressors). In our laboratory, baboons have been used to study DNA vaccine strategies using new cationic liposome formulations and granulocyte macrophage-colony stimulating factor and B7-2 as genetic adjuvants. The results demonstrate the value of using baboons as an animal model of AIDS pathogenesis and vaccine development.

Antibody and cellular immune responses in breakthrough infection subjects after HIV type 1 glycoprotein 120 vaccination.

HIV-specific antibodies and CD8+ T cell antiviral responses were evaluated in three human immunodeficiency virus 1 (HIV-1) gp120 vaccine recipients who later became infected with HIV-1. Titers of neutralizing antibody to the HIV-1(SF2) vaccine isolate were boosted, but titers of antibody to the autologous infecting viruses were never high and required at least 6 months after HIV infection to develop. Similarly, a marginal noncytotoxic CD8+ T cell antiviral response was observed only in one of the three vaccinees 3 months after HIV-1 infection. The infecting virus isolates had several amino acid substitutions in the HIV-1 envelope V3 region but were similar to other regional HIV-1 clade B isolates. Viral loads were similar to those of other HIV-1-infected individuals who had not been vaccinated and transient CD4+ T cell declines were observed in each person, suggesting that the vaccine was not effective at controlling these prognostic markers early in infection.

Suppression of human immunodeficiency virus type 1 replication by a soluble factor produced by CD8+ lymphocytes from HIV-2-infected baboons.

Human immunodeficiency virus type 2 (HIV-2)-infected baboons (Papio cynocephalus) provide a valuable animal model for the study of acquired immunodefidency syndrome (AIDS) pathogenesis since many features of disease progression resemble HIV-1-infection of humans. In some HIV-2-infected baboons that are clinically healthy, a CD8+ cell antiviral response, that is partly mediated by a soluble factor, controls viral replication in vitro. In the present study, we demonstrate that CD8+ cells derived from HIV-2-infected baboon peripheral blood, lymph nodes, adenoids and tonsils had antiviral activity in co-cultures of CD8+ and CD4+ cells that inversely correlates with viral load. A soluble factor was found to be active against the chemokine-resistant, syncytium-inducing HIV-1SF2 and HIV-1SF33 isolates and was relatively heat stable at 100 degrees C for 10 min. Moreover, inhibition of the transcription from the long terminal repeat of HIV-1 was observed in 1G5 cells after activation with phorbol 12-myristate 13-acetate. Therefore, the soluble suppressing activity of CD8+ cells in HIV-2-infected baboons may be analogous to the CD8+ cell antiviral factor described in human HIV-infected asymptomatic people.

Human immunodeficiency virus-2 infection in baboons is an animal model for human immunodeficiency virus pathogenesis in humans.

OBJECTIVE: To assess disease progression in baboons (Papio cynocephalus) that were infected with two human immunodeficiency virus-2 (HIV-2) isolates. METHODS: Eight baboons were inoculated intravenously with either HIV-2UC2 or HIV-2UC14 and were followed for a 2- to 7-year period of observation. RESULTS: Six of 8 baboons showed lymphadenopathy and other signs of HIV-related disease, 3 of 8 baboons had an acute phase CD4+ T-cell decline, and 2 of 5 baboons infected with the HIV-2UC2 isolate progressed to an acquired immunodeficiency syndrome-like disease. Human immunodeficiency virus-2-specific pathology in lymphatic tissues included follicular lysis, vascular proliferation, and lymphoid depletion. Both neutralizing antibodies and a CD8+ T-cell antiviral response were associated with resistance to disease. CONCLUSIONS: Disease progression and the development of acquired immunodeficiency syndrome in HIV-2-infected baboons have similarities to human HIV infections.

Transient virus infection and pathogenesis of a new HIV type 2 isolate, UC12, in baboons.

We have previously shown that baboons (Papio cynocephalus) can be persistently infected with HIV-2 and some baboons progress to an AIDS-like disease with a CD4+ T cell decline, cachexia, alopecia, and Kaposi's sarcoma-like fibromatosis. In this study, we found that a new virus isolate, HIV-2UC12, replicated to high levels in baboon peripheral blood mononuclear cells (PBMCs) in vitro. Three baboons were subsequently inoculated and had plasma viral RNA loads that peaked between 15,000 and 7000 copies/ml at 2 weeks postinfection. Virus was isolated from the PBMCs for up to 6 months. Although PBMCs were subsequently virus culture negative, virus could be recovered from the spleen, lymph nodes, and tonsils, indicating that HIV-2 was sequestered within these lymphoid tissues. HIV-2-associated pathology included follicular lysis, vascular proliferation, and lymphoid depletion. This study indicated that HIV-2UC12 infection in baboons can cause HIV-associated pathological abnormalities within the lymphatic tissues and that the high level of HIV-2UC12 replication in vitro was not predictive of replication in vivo.

Superinfection with human immunodeficiency virus type 2 can reactivate virus production in baboons but is contained by a CD8 T cell antiviral response.

An animal model was used to assess whether resistance to superinfection by human immunodeficiency virus (HIV) can exist in vivo. Asymptomatic baboons (Papio cynocephalus), previously infected with HIV-2, were first challenged with homologous virus (HIV-2UC2 or HIV-2UC14) and later with heterologous virus (HIV-2UC12). After both virus inoculations, either resistance to viral infection or a transient viremia was observed. The original virus was recovered in 3 baboons, suggesting that reactivation of a latent infection occurred on heterologous challenge and that HIV-2 superinfection is blocked by processes established during prior infection. Antibody titers measured by ELISA and virus neutralization remained at low levels. However, suppression of HIV-1 replication was observed with CD8 T cells and filtered cell culture supernatants. The soluble factor involved was not a beta-chemokine. This resistance to HIV superinfection appears to be mediated at least in part by CD8 T cells that suppress virus production.

CD8+ cells from HIV-2-infected baboons control HIV replication.

OBJECTIVE: To analyze the CD8+ cell antiviral immune response in HIV-2-infected baboons. DESIGN: Baboons were infected with clinical isolates of HIV-2, CD8+ cells were isolated from phytohemagglutinin (PHA)-stimulated baboon peripheral blood mononuclear cells (PBMC). These cells were cultured with PHA-stimulated CD4+ cells acutely infected with HIV-2 at several CD8+:CD4+ cell ratios. Control of HIV-2 replication was determined by comparing peak levels of HIV-2 replication in fluids from CD8+:CD4+ cell cocultures with those in fluids from infected CD4+ cells cultured alone. RESULTS: CD8+ cells from HIV-2-infected baboons inhibited HIV-2 replication in acutely infected autologous CD4+ cells to a significantly greater extent than did CD8+ cells from uninfected baboons (P = 0.0001). At the beginning of the acute phase of HIV-2 infection, CD8+ cells showed either a transient reduction or loss in the antiviral activity. In some cases the CD8+ cell response enhanced HIV-2 replication. Subsequently, the strength of the CD8+ cell antiviral activity increased concomitant with a decrease in the HIV-2 load in the PBMC. Suppression of HIV replication could be demonstrated with filtered fluid from CD8+ cells. Other studies indicated that infected CD4+ cells are lost during coculture of CD8+ cells with infected CD4+ cells. CONCLUSIONS: CD8+ cells of HIV-2-infected baboons develop substantial anti-HIV-2 activity following HIV-2 infection, which may account in part for the low frequency of pathogenesis in HIV-2-infected baboons. Studies to elucidate the mechanism of this CD8+ cell antiviral activity suggest that it is mediated in part by a soluble antiviral factor, but primarily in association with the loss of infected CD4+ cells.

CD8+ cells from asymptomatic human immunodeficiency virus-infected individuals suppress superinfection of their peripheral blood mononuclear cells.

Most human immunodeficiency virus (HIV)-infected individuals show evidence of infection by only one strain of the virus despite possible frequent contact with multiple strains. The reason(s) for the emergence of a dominant strain of virus in HIV-infected people and the mechanism(s) which prevent other strains from establishing an infection is not known. In the present study, we demonstrate that peripheral blood mononuclear cells (PBMC) of asymptomatic HIV-infected individuals can resist productive infection by HIV-1 and HIV-2 strains. Although the PBMC of these individuals are resistant to superinfection, their CD4+ cells are susceptible to infection. Moreover, two weeks after infection of their PBMC in culture, the superinfecting virus can be recovered from isolated CD4+ cells. When CD8+ cells from asymptomatic individuals are added to the superinfected CD4+ cells, replication of the exogenously introduced virus is inhibited. In contrast, PBMC from individuals who have progressed to disease (Progressors) do not resist superinfection and their CD8+ cells do not showthe antiviral activity which controls productive HIV infection. These findings suggest that CD8+ cells suppressing HIV replication in infected individuals may be critical in preventing the establishment of infection by other strains of HIV by blocking virus replication.

Plasmodium falciparum: gp195 tripeptide repeat-specific monoclonal antibody inhibits parasite growth in vitro.

Seven monoclonal antibodies (mAbs) were produced to the precursor of the merozoite surface antigens (MSA-1 or gp195) using the Plasmodium falciparum Uganda-Palo Alto isolate. Three mAbs (CE2, DB8, and EB2) reacted with epitopes on the 83-kDa N-terminal processing fragment by immunoprecipitation of radiolabeled proteins and in immunoblots of native and recombinant proteins. Three other mAbs (BC9, AG5, and AD9) reacted with epitopes on the 42- and 19-kDa C-terminal processing fragments while one remaining mAb (24A1.7) reacted with only 150- and 110-kDa intermediate processing fragments. Epitopes were mapped to either conserved or dimorphic regions of the expressed protein when parasite isolates with known MSA-1 alleles were examined by indirect immunofluorescence. Moreover, one mAb (CE2), specific for the variable tripeptide repeat region SAQ(SGT)5, was growth inhibitory for P. falciparum in vitro. Growth inhibition by the mAb was concentration dependent and its parasite-neutralizing properties were not enhanced when used in combination with other gp195-specific mAbs. These results may be useful in the elucidation of biological variation of field isolates and in the definition of immunologically relevant epitopes in a gp195-based malaria vaccine.

Molecular cloning of the human immunodeficiency virus subtype 2 strain HIV-2UC2.

An infectious molecular clone was derived from the HIV-2UC2 isolate that previously was found to persistently infect and induce an AIDS-like disease syndrome in baboons. The molecularly cloned virus (HIV-2UC2mc) showed in vitro properties similar to those of the parental isolate with regard to T-cell tropism, cytopathicity, and the ability to infect primary baboon PBMC. Nevertheless, when inoculated into two baboons, the cloned virus showed a limited ability to replicate in these animals. DNA sequence analysis revealed a defective vpr gene in the UC2mc as well as in the pathogenic parental UC2 strain. Thus, the vpr gene is not required for the induction of disease in baboons. The attenuated infectious molecular clone of UC2 should be useful for future studies designed to map the genetic determinants of HIV-2 pathogenesis in the baboon model and to evaluate vaccine strategies.

Antiviral activity of Hawaiian medicinal plants against human immunodeficiency Virus Type-1 (HIV-1).

Hawaiian medicinal plants commonly used for the treatment of a variety of infections were screened for antiviral activity against human immunodeficiency virus type 1 (HIV-1). Sixty-one extracts derived from seventeen plants were tested for selective viral growth inhibition using the LAI (HTLV-IIIB) isolate. The greatest degree of antiviral activity was observed with aqueous extracts made from the bark of Eugenia malaccensis (L.) and the leaves of Pluchea indica (Less.) which had antiviral selectivity indices (50% cytotoxic concentration/50% effective antiviral concentration) of 109 and 94, respectively. These and other extracts conferred 100% cell protection against viral cytopathic effect when compared with control samples. Methanol and water extracts made from the Pipturus albidus (Gray) leaves and bark also achieved a high selective inhibition of virus replication with very low cytotoxicity. Plant extracts made from Aleurites moluccana (Willd.), Psychotria hawaiiensis (Gray), Clermontia aborescens (Mann), and Scaevola sericea (Forst.) also showed antiviral activity. These data provide a rationale for the characterization of antiviral natural products from these plants and related plant species.

Anti-microbial activity and anti-complement activity of extracts obtained from selected Hawaiian medicinal plants.

Selected plants having a history of use in Polynesian traditional medicine for the treatment of infectious disease were investigated for anti-viral, anti-fungal and anti-bacterial activity in vitro. Extracts from Scaevola sericea, Psychotria hawaiiensis, Pipturus albidus and Eugenia malaccensis showed selective anti-viral activity against Herpes Simplex Virus-1 and 2 and Vesicular Stomatitis Virus. Aleurites moluccana extracts showed anti-bacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa, while Pipturus albidus and Eugenia malaccensis extracts showed growth inhibition of Staphylococcus aureus and Streptococcus pyogenes. Psychotria hawaiiensis and Solanum niger inhibited growth of the fungi Microsporum canis, Trichophyton rubrum and Epidermophyton floccosum, while Ipomoea sp., Pipturus albidus, Scaevola sericea, Eugenia malaccensis, Piper methysticum, Barringtonia asiatica and Adansonia digitata extracts showed anti-fungal activity to a lesser extent. Eugenia malaccensis was also found to inhibit the classical pathway of complement suggesting that an immunological basis for its in vivo activity was identified. This study has confirmed some of the ethnobotanical reports of Hawaiian medicinal plants having curative properties against infections using biological assays in vitro.

Reduction of disulfide bonds in Plasmodium falciparum gp195 abolishes the production of growth-inhibitory antibodies.

The role of disulfide-dependent protein conformation of the 195,000 kDa Plasmodium falciparum merozoite surface glycoprotein in the induction of biologically active antibodies was investigated. Serum samples from rabbits immunized with native gp195 had a mean ELISA titre of 1/560,000 and a mean in vitro parasite growth inhibition of 80%. In contrast, serum samples from rabbits immunized with reduced and alkylated gp195 had a mean antibody titre of 1/23,100 and did not inhibit parasite growth. These results indicate that the native structure of gp195 is essential for antigenicity, immunogenicity and induction of growth-inhibitory antibodies. Therefore, effective recombinant gp195-based vaccines may require the production of properly folded molecules resembling the native conformation.

Three matrix metalloproteinases form a non-covalent association with the rhoptry-associated protein-1 of Plasmodium falciparum.

During the characterization of malaria vaccine candidate proteins, three metalloproteinases having a molecular mass of 220, 95 and 70 kDa were found to be co-isolated with the rhoptry-associated protein-1 (RAP-1) complex, but not with RAP-3 or gp195. These enzymes were also found in detergent extracts of saponin-lysed Plasmodium falciparum. Of nine proteinase inhibitors tested, only EDTA was found to abrogate activity. Dose-dependent curves were determined for several metal ions and cobalt was found to synergistically enhance enzyme activity. The gelatinases were immunoprecipitated with monospecific polyclonal antisera to macrophage and fibroblast gelatinase; however, these sera did not react with intracellular parasites by indirect immunofluorescence. These results indicate that the matrix metalloproteinases co-isolated with RAP-1 originate from human serum used to cultivate P. falciparum in vitro.