Serum-free transient protein production system based on adenoviral vector and PER.C6 technology: high yield and preserved bioactivity.

Stable E1 transformed cells, like PER.C6, are able to grow at scale and to high cell densities. E1-deleted adenoviruses replicate to high titer in PER.C6 cells whereas subsequent deletion of E2A from the vector results in absence of replication in PER.C6 cells and drastically lowers the expression of adenovirus proteins in such cells. We therefore considered the use of an DeltaE1/DeltaE2 type 5 vector (Ad5) to deliver genes to PER.C6 cells growing in suspension with the aim to achieve high protein yield. To evaluate the utility of this system we constructed DeltaE1/DeltaE2 vector carrying different classes of protein, that is, the gene coding for spike protein derived from the Coronavirus causing severe acute respiratory syndrome (SARS-CoV), a gene coding for the SARS-CoV receptor or the genes coding for an antibody shown to bind and neutralize SARS-CoV (SARS-AB). The DeltaE1/DeltaE2A-vector backbones were rescued on a PER.C6 cell line engineered to constitutively over express the Ad5 E2A protein. Exposure of PER.C6 cells to low amounts (30 vp/cell) of DeltaE1/DeltaE2 vectors resulted in highly efficient (>80%) transduction of PER.C6 cells growing in suspension. The efficient cell transduction resulted in high protein yield (up to 60 picogram/cell/day) in a 4 day batch production protocol. FACS and ELISA assays demonstrated the biological activity of the transiently produced proteins. We therefore conclude that DeltaE1/DeltaE2 vectors in combination with the PER.C6 technology may provide a viable answer to the increasing demand for high quality, high yield recombinant protein.

Novel replication-incompetent adenoviral B-group vectors: high vector stability and yield in PER.C6 cells.

Adenoviral vectors based on adenovirus type 35 (rAd35) have the advantage of low natural vector immunity and induce strong, insert-specific T- and B-cell responses, making them prime-candidate vaccine carriers. However, severe vector-genome instability of E1-deleted rAd35 vectors was observed, hampering universal use. The instability of E1-deleted rAd35 vector proved to be caused by low pIX expression induced by removal of the pIX promoter, which was located in the E1B region of B-group viruses. Reinsertion of a minimal pIX promoter resulted in stable vectors able to harbour large DNA inserts (> 5 kb). In addition, it is shown that replacement of the E4-Orf6 region of Ad35 by the E4-Orf6 region of Ad5 resulted in successful propagation of an E1-deleted rAd35 vector on existing E1-complementing cell lines, such as PER.C6 cells. The ability to produce these carriers on PER.C6 contributes significantly to the scale of manufacturing of rAd35-based vaccines. Next, a stable rAd35 vaccine was generated carrying Mycobacterium tuberculosis antigens Ag85A, Ag85B and TB10.4. The antigens were fused directly, resulting in expression of a single polyprotein. This vaccine induced dose-dependent CD4+ and CD8+ T-cell responses against multiple antigens in mice. It is concluded that the described improvements to the rAd35 vector contribute significantly to the further development of rAd35 carriers for mass-vaccination programmes for diseases such as tuberculosis, AIDS and malaria.

Immunogenicity and protection of a recombinant human adenovirus serotype 35-based malaria vaccine against Plasmodium yoelii in mice.

Given the promise of recombinant adenovirus type 5 (rAd5) as a malaria vaccine carrier in preclinical models, we evaluated the potency of rAd35 coding for Plasmodium yoelii circumsporozoite protein (rAd35PyCS). We chose rAd35 since a survey with serum samples from African subjects demonstrated that human Ad35 has a much lower seroprevalence of 20% and a much lower geometric mean neutralizing antibody titer (GMT) of 48 compared to Ad5 (seroprevalence, 85%; GMT, 1,261) in countries with a high malaria incidence. We also demonstrated that immunization with rAd35PyCS induced a dose-dependent and potent, CS-specific CD8(+) cellular and humoral immune response and conferred significant inhibition (92 to 94%) of liver infection upon high-dose sporozoite challenge. Furthermore, we showed that in mice carrying neutralizing antibody activity against Ad5, mimicking a human situation, CS-specific T- and B-cell responses were significantly dampened after rAd5PyCS vaccination, resulting in loss of inhibition of liver infection upon sporozoite challenge. In contrast, rAd35 vaccine was as potent in naive mice as in Ad5-preimmunized mice. Finally, we showed that heterologous rAd35-rAd5 prime-boost regimens were more potent than rAd35-rAd35 because of induction of anti-Ad35 antibodies after rAd35 priming. The latter data provide a further rationale for developing rAd prime-boost regimens but indicate that priming and boosting Ad vectors must be immunologically distinct and also should be distinct from Ad5. Collectively, the data presented warrant further development of rAd35-based vaccines against human malaria.

Characteristics of a pathogenic molecular clone of an end-stage serum-derived variant of simian immunodeficiency virus (SIV(F359)).

End-stage simian immunodeficiency virus (SIV) isolates are suggested to be the most fit of the evolved virulent variants that precipitate the progression to AIDS. To determine if there were common characteristics of end-stage variants which emerge from accelerated cases of AIDS, a molecular clone was derived directly from serum following in vivo selection of a highly virulent SIV isolate obtained by serial end-stage passage in rhesus monkeys (Macaca mulatta). This dominant variant caused a marked cytopathic effect and replicated to very high levels in activated but not resting peripheral blood lymphocytes. Furthermore, although this clone infected but did not replicate to detectable levels in rhesus monocyte-derived macrophages, these cells were able to transmit infection to autologous T cells upon contact. Interestingly, although at low doses this end-stage variant did not use any of the known coreceptors except CCR5, it was able to infect and replicate in human peripheral blood mononuclear cells homozygous for the Delta 32 deletion of CCR5, suggesting the use of a novel coreceptor. It represents the first pathogenic molecular clone of SIV derived from viral RNA in serum and provides evidence that not only the genetic but also the biological characteristics acquired by highly fit late-stage disease variants may be distinct in different hosts.

The rate of progression to AIDS is independent of virus dose in simian immunodeficiency virus-infected macaques.

Of the viral factors that are proposed to influence the rate of progression to AIDS, the role of infectious dose remains unresolved. Intravenous infection of outbred Macaca mulatta with various doses of simian immunodeficiency virus isolate 8980 (SIV(8980)) revealed an endpoint from which an infectious dose 50 (ID(50)) was defined. In the six infected animals, the time to develop AIDS was variable with a spectrum of rapid, intermediate and slow progressors. High and sustained plasma viraemia with marked loss of CD4(+) T-cells was a distinguishing feature between rapid versus intermediate and slow progressors. Animals that received the highest doses did not develop the highest sustained viral loads, nor did they progress more rapidly to disease. Similarly, animals infected with lower doses did not uniformly develop lower viral loads or progress more slowly to AIDS. Furthermore, compiled data from more than 21 animals infected with different doses of the same virus administered by the same route failed to reveal any correlation of infectious dose with survival. Indeed, host factors of these outbred animals, rather than dose of the initial inoculum, were probably an important factor influencing the rate of disease progression in each individual animal. Comparison of animals infected with SIV(B670), from which SIV(8980) was derived, revealed marked differences in disease progression. Clearly, although dose did not influence viral loads nor disease progression, the virulence of the initial inoculum was a major determinant of the rate of progression to AIDS.

A strategy for cloning infectious molecular clones of retroviruses from serum or plasma.

To enable biological characterisation of lentiviral variants which emerge during infection and development of AIDS, a method was developed to construct molecular clones from circulating simian immunodeficiency virus (SIV) particles present in as little as 20 microl of serum from infected rhesus monkeys. This technique uses a long distance RT-PCR method optimised for the amplification of partly overlapping 5-kb SIV (half genome) amplimers. Ligation of the genome halves resulted in the construction of full-length clones which, after transfection, were able to replicate well in rhesus peripheral blood mononuclear cells (PBMCs) and in various human T-cell lines inducing syncytia. In addition to the study of molecular cloned virus quasispecies emerging in circulation as a result of immune escape, this method may also be applied to obtain entire genes or full-length molecular clones. These clones may be present in other extracellular body fluids such as urine, saliva, tears, lymph, and bronchial or cerebral spinal fluid. Genes amplified in this way can be inserted quickly in new recombinant expression vectors and may then be applied for DNA vaccination approaches.

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 degrees-42 degrees 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.

Vaccine protection and reduced virus load from heterologous macaque-propagated SIV challenge.

The efficacy of vaccine protection afforded by live attenuated vaccines was tested by heterologous SIVtno8980 challenge following successful protection against homologous SIVmac32H challenge. Animals immunized with the attenuated SIVmacBK28 molecular clone were asymptomatic and virus isolation negative by quantitative virus isolation prior to challenge. Two groups of four animals previously immunized 5 years and 4 months (respectively) were challenged with 100 MID50 of SIVtno8980, as was a third group of four naive controls. All control animals that were challenged developed high levels of plasma antigenemia within 2 weeks of challenge and developed rapid Th/m cell loss whereas vaccinated animals did not. Quantitative virus isolation from peripheral blood mononuclear cells revealed that one of four animals in each group became virus isolation positive but that the virus load in the two vaccinated animals was markedly lower than in nonvaccinated controls. Studies are underway to determine the duration and immunological correlates of protection from AIDS.

Lymphoproliferative disorders developing after transplantation and their relation to simian T-cell leukemia virus infection.

In this report the role of the HTLV-1-like simian T-cell leukemia virus (STLV) during the development of posttransplantation lymphoproliferative disorders (PTLPD) is described. To prevent rejection of an allogeneic transplant in 12 rhesus monkeys cyclosporin A (CyA), prednisone, and/or lymphocyte-specific monoclonal antibodies were used for immunosuppression. Seven monkeys died during the experiment between 22 and 179 days postoperatively. At autopsy in 4 monkeys PTLPD were found. In each case, STLV provirus was acquired during the experiment, either from the blood transfusions or allograft donors. Seroconversion of anti-STLV titers occurred in 3 monkeys. However, Southern blot analysis showed the presence of STLV provirus at the DNA level in all PTLP tissues. PTLPD morphology and phenotype varied significantly. In conclusion, for the first time the oncogenic potential of STLV is identified in a rhesus monkey transplantation model. Moreover, the importance of screening blood and organ donors for HTLV-1 must be emphasized.

Molecular cloning of the gene for the E1 alpha subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae.

The E1 alpha and E1 beta subunits of the pyruvate dehydrogenase complex from the yeast Saccharomyces cerevisiae were purified. Antibodies raised against these subunits were used to clone the corresponding genes from a genomic yeast DNA library in the expression vector lambda gt11. The gene encoding the E1 alpha subunit was unique and localized on a 1.7-kb HindIII fragment from chromosome V. The identify of the gene was confirmed in two ways. (a) Expression of the gene in Escherichia coli produced a protein that reacted with the anti-E1 alpha serum. (b) Gene replacement at the 1.7-kb HindIII fragment abolished both pyruvate dehydrogenase activity and the production of proteins reacting with anti-E1 alpha serum in haploid cells. In addition, the 1.7-kb HindIII fragment hybridized to a set of oligonucleotides derived from amino acid sequences from the N-terminal and central regions of the human E1 alpha peptide. We propose to call the gene encoding the E1 alpha subunit of the yeast pyruvate dehydrogenase complex PDA1. Screening of the lambda gt11 library using the anti-E1 beta serum resulted in the reisolation of the RAP1 gene, which was located on chromosome XIV.