The pathogenesis of AIDS: lessons from the SIV-Macaque model

In the mid-eighties it became apparent that a human retrovirus of the lentivirus subfamily, later designated human immunodeficiency virus type I (HIV-l), was the etiological agent of the acquired immune deficiency syndrome (AIDS). This disease was characterised by unusual opportunistic infections, neurologic abnormalities, gastrointestinal disorders and malignancies due to an insidious decay of the immune system. The urgent need to gain insight in certain aspects of the pathobiology of this infection demanded relevant animal models. As a consequence of this need the search for similar lentivimses present in other animal species intensified. Several lentivimses have been identified to induce AIDS-like disease in a vruiety of animals , however, only simian immunodeficiency virus (SIV) and feline inununodeficiency virus (FIV), wWcb cause AIDS-like symptoms in macaques and cats, respectively, have been commonly used as animal models. In this chapter an overview of the molecular biology of SIV and the utilisation of the SIV -macaque model for AIDS research is given. In prulicular, the use of molecular clones of SIV to elucidate the pathogenesis of AIDS is described

Direct Amplification and Cloning of Up to 5-kb Lentivirus Genomes from Serum

To produce large cDNA strands from biological samples containing limited numbers of template molecules, it may be necessary to minimize both nonspecific primer attachment in first-strand synthesis and secondary structure in RNA molecules. Failure to do so could result in the accumulation of shortened cDNA strands and therefore may reduce the yield of large cDNA molecules, sometimes below detection level. We show that 5.0-kb cDNA fragments can be generated from simian immunodeficiency virus RNA in a specific reverse transcription (RT)-PCR by increasing the stringency of the primer-annealing conditions, followed by the elimination of excess free primer. Since this method utilizes a relatively long primer in the first-strand cDNA synthesis, it is possible to heat-denature the nonspecific RNA/primer complexes and RNA secondary structure without dissociating the primer from the specific template. In contrast to classic RT assays, in which an excess of primer is annealed to denatured RNA just prior to and during reverse transcription at relative low temperatures (37°–42°C), this method eliminates false priming. To optimize the yield and fidelity of full-length cDNA molecules, two PCR amplifications are first performed using both Taq and Pfu polymerase, followed by Pfu alone in the second amplification

Myeloid and plasmacytoid dendritic cells are susceptible to recombinant adenovirus vectors and stimulate polyfunctional memory T cell responses

Although replication-incompetent recombinant adenovirus (rAd) type 5 is a potent vaccine vector for stimulating T and B cell responses, high seroprevalence of adenovirus type 5 (Ad5) within human populations may limit its clinical utility. Therefore, alternative adenovirus serotypes have been studied as vaccine vectors. In this study, we characterized the ability of rAd5 and rAd35 to infect and induce maturation of human CD11c(+) myeloid dendritic cells (MDCs) and CD123(+) plasmacytoid dendritic cells (PDCs), and their ability to stimulate Ag-specific T cells. Both MDCs and PDCs were found to express the primary receptor for Ad35 (CD46) but not Ad5 (coxsackie-adenovirus receptor; CAR). Both dendritic cell (DC) subsets were also more susceptible to rAd35 than to rAd5. MDCs were more susceptible to both rAd35 and rAd5 than were PDCs. Whereas rAd35 used CD46 for entry into DCs, entry of rAd5 may be through a CAR-independent pathway. Exposure to rAd35 but not rAd5 induced high levels of IFN-alpha in PDCs and phenotypic differentiation in both DC subsets. MDCs and PDCs exposed to either rAd5 or rAd35 encoding for CMV pp65 were able to present pp65 and activate CMV-specific memory CD8(+) and CD4(+) T cells in a dose-dependent manner, but MDCs stimulated the highest frequencies of pp65-specific T cells. Responding T cells expressed multiple functions including degranulation (CD107a surface mobilization) and production of IFN-gamma, IL-2, TNF-alpha, and MIP-1beta. Thus, the ability of rAd35 to naturally target important DC subsets, induce their maturation, and appropriately present Ag to T cells may herald greater in vivo immunogenicity than has been observed with rAd

Comparative characterization of hexose transporters of Plasmodium knowlesi, Plasmodium yoelii and Toxoplasma gondii highlights functional differences within the apicomplexan family.

Chemotherapy of apicomplexan parasites is limited by emerging drug resistance or lack of novel targets. PfHT1, the Plasmodium falciparum hexose transporter 1, is a promising new drug target because asexual-stage malarial parasites depend wholly on glucose for energy. We have performed a comparative functional characterization of PfHT1 and hexose transporters of the simian malarial parasite P. knowlesi (PkHT1), the rodent parasite P. yoelii (PyHT1) and the human apicomplexan parasite Toxoplasma gondii ( T. gondii glucose transporter 1, TgGT1). PkHT1 and PyHT1 share >70% amino acid identity with PfHT1, while TgGT1 is more divergent (37.2% identity). All transporters mediate uptake of D-glucose and D-fructose. PyHT1 has an affinity for glucose ( K (m) approximately 0.12 mM) that is higher than that for PkHT1 ( K (m) approximately 0.67 mM) or PfHT1 ( K (m) approximately 1 mM). TgGT1 is highly temperature dependent (the Q (10) value, the fold change in activity for a 10 degrees C change in temperature, was >7) compared with Plasmodium transporters ( Q (10), 1.5-2.5), and overall has the highest affinity for glucose ( K (m) approximately 30 microM). Using active analogues in competition for glucose uptake, experiments show that hydroxyl groups at the C-3, C-4 and C-6 positions are important in interacting with PkHT1, PyHT1 and TgGT1. This study defines models useful to study the biology of apicomplexan hexose permeation pathways, as well as contributing to drug development

High-level expression from two independent expression cassettes in replication-incompetent adenovirus type 35 vector

Replication-incompetent adenovirus type 35 (rAd35) represents a potent vaccine carrier that elicits strong, antigen-specific T- and B-cell responses in diverse preclinical models. Moreover, Ad35 is rare in human populations, resulting in the absence of neutralizing antibodies against this carrier, in contrast to the commonly used rAd5. Therefore, rAd35 is being investigated as a vaccine carrier for a number of diseases for which an effective vaccine is needed, including malaria, AIDS and tuberculosis, However, it can be perceived that effective immunization will require insertion of multiple antigens into adenoviral vectors. We therefore wanted to create rAd35 vectors carrying double expression cassettes, to expand within one vector the number of insertion sites for foreign DNA encoding antigenic proteins. We show that it is possible to generate rAd35 vectors carrying two cytomegalovirus promoter-driven expression cassettes, provided that the polyadenylation signals in each expression cassette are not identical. We demonstrate excellent rAd35 vector stability and show that expression of a transgene is not influenced by the presence of a second expression cassette. Moreover, by using two model vaccine antigens, i.e. the human immunodeficiency virus-derived Env-gp120 protein and the Plasmodium falciparum-derived circumsporozoite protein, we demonstrate that potent T- and B-cell responses are induced to both antigens expressed from a single vector. Such rAd35 vectors thus expand the utility of rAd35 vaccine carriers for the development of vaccines against, for example, malaria, AIDS and tuberculosi

Generation of a novel replication-incompetent adenoviral vector derived from human adenovirus type 49: manufacture on PER.C6 cells, tropism and immunogenicity

Recombinant adenoviral vectors based on type 5 (rAd5) show great promise as a vaccine carrier. However, neutralizing activity against Ad5 is prevalent and high-titred among human populations, and significantly dampens Ad5-based vaccine modalities. The generation of alternative adenoviral vectors with low seroprevalence thus receives much research attention. Here, it is shown that a member from human adenovirus subgroup D, i.e. Ad49, does not cross-react with Ad5 neutralizing activity, making it a candidate serotype for vector development. Therefore, a plasmid system that allows formation of replication-incompetent adenovirus serotype 49 vaccine vectors (rAd49) was constructed and it was demonstrated that rAd49 can be successfully propagated to high titres on existing Ad5.E1-complementing cell lines such as PER.C6. Using an rAd49 vector carrying the luciferase marker gene, detailed seroprevalence studies were performed, demonstrating that rAd49 has low seroprevalence and neutralizing antibody titres worldwide. Also, we have initiated rAd49 vector receptor usage suggesting that rAd49 utilizes hCD46 as a cellular receptor. Finally, the immunogenicity of the rAd49 vector was assessed and it was shown that an rAd49.SIVGag vaccine induces strong anti-SIVGag CD8+ T-lymphocytes in naïve mice, albeit less than an rAd5.SIVGag vaccine. However, in mice with high anti-Ad5 immunity the rAd5.SIVGag vaccine was severely blunted, whereas the anti-SIVGag response was not significantly suppressed using the rAd49.SIVGag vaccine. These data demonstrate the potential of a replication deficient human group D adenoviral vector for vaccination purpose

Impact of Recombinant Adenovirus Serotype 35 Priming versus Boosting of a Plasmodium falciparum Protein: Characterization of T- and B-Cell Responses to Liver-Stage Antigen 1▿

Prime-boost vaccination regimens with heterologous antigen delivery systems have indicated that redirection of the immune response is feasible. We showed earlier that T-cell responses to circumsporozoite (CS) protein improved significantly when the protein is primed with recombinant adenovirus serotype 35 coding for CS (rAd35.CS). The current study was designed to answer the question whether such an effect can be extended to liver-stage antigens (LSA) of Plasmodium falciparum such as LSA-1. Studies with mice have demonstrated that the LSA-1 protein induces strong antibody response but a weak T-cell immunity. We first identified T-cell epitopes in LSA-1 by use of intracellular gamma interferon (IFN-γ) staining and confirmed these epitopes by means of enzyme-linked immunospot assay and pentamer staining. We show that a single immunization with rAd35.LSA-1 induced a strong antigen-specific IFN-γ CD8+ T-cell response but no measurable antibody response. In contrast, vaccinations with the adjuvanted recombinant LSA-1 protein induced remarkably low cellular responses but strong antibody responses. Finally, both priming and boosting of the adjuvanted protein by rAd35 resulted in enhanced T-cell responses without impairing the level of antibody responses induced by the protein immunizations alone. Furthermore, the incorporation of rAd35 in the vaccination schedule led to a skewing of LSA-1-specific antibody responses toward a Th1-type immune response. Our results show the ability of rAd35 to induce potent T-cell immunity in combination with protein in a prime-boost schedule without impairing the B-cell response

Increased immunogenicity of recombinant Ad35-based malaria vaccine through formulation with aluminium phosphate adjuvant

Previously, we have shown the potency of recombinant Adenovirus serotype 35 viral vaccines (rAd35) to induce strong immune response against the circumsporozoite protein (CS) of the plasmodium parasite. To further optimize immunogenicity of Ad35-based malaria vaccines we formulated rAd35.CS vaccine with aluminium phosphate adjuvant (AlPO(4)). In contrast to the conventional protein based vaccines no absorption to aluminium adjuvant was observed and rAd35 viral in vitro infectivity in mammalian cells was preserved. Immunization with Ad35.CS formulated with AlPO(4) resulted in significantly higher CS specific T and B cell responses in mice upon either single or prime-boost vaccination regimens as compared to rAd35.CS alone. With these results we report for the first time the feasibility of using an AlPO(4) adjuvant to increase the potency of a live adenovirus serotype 35-based vaccin