Measurement of Antigen Expression

In the case of live-attenuated viral vaccines, viral antigens are expressed in infected cells with limited virus replication, and without the pathogenic effects of the parental live virus. The viral antigens may be detected and measured by using specific polyclonal antisera or monoclonal antibodies (MAbs) for immunostaining of fixed cells or Western blotting of extracted proteins. Such techniques were less frequently used on the now well-established viral vaccines such as live-attenuated polio vaccine, but are increasingly applied to novel vaccines based on live recombinant viral vectors encoding and expressing heterologous antigens. For example, poxviruses have large DNA genomes with a high capacity for the insertion of heterologous DNA sequences encoding and expressing single/multiple heterologous antigen(s). In particular, vaccinia virus and avian poxviruses (canarypox and fowlpox) are considered as promising candidates for the development of vaccines for use in humans and in the veterinary field [5-8]. Attenuated strains of vaccinia virus - for example modified vaccinia virus Ankara (MVA) and NYVAC, canarypox virus (e.g., ALVAC) and fowlpox virus (e.g., FP9 and TROVAC) - have been isolated that replicate in chicken embryo fibroblasts (CEF), but not in human cells, and these are now being widely developed as recombinant viral vector vaccines [6, 9-11, 13]. For example, recombinant poxviruses encoding various antigens of disease-causing viruses, including influenza virus, parainfluenza virus, measles virus, respiratory syncytial virus, dengue virus, Japanese encephalitis virus, human immunodeficiency virus-1 (HIV-1) and P. falciparum (malaria) antigens, have been characterized in vitro [10-14, 94]. Despite abortive replication in human cells, the heterologous genes are still expressed; the heterologous antigens are produced and have been found to stimulate immune responses. Examples are the development of antigen-specific T cells and antibodies in animal model systems and in humans.

Since mutations (including deletions) within recombinant MVA or ALVAC vector vaccine DNA can occur at high passage levels, it is considered essential to monitor the consistency of heterologous antigen expression to ensure the batch-to-batch stability of potency [15]. Such monitoring requires the development of validated potency assays to determine expression levels, and concurrently the development of stable, well-characterized reference materials to permit comparison of results among assays and different vaccine batches. For ALVAC-HIV, a recombinant ALVAC viral vector that expresses the HIV-1 p24 and gp120 antigens, potency assays have been based on: (1) HIV-1 gene expression in CEF (immunoplaque assay); (2) HIV-1 gene expression in the human HeLa cell line (Fluorescence Activated Cell Sorter, FACS, analysis); and (3) immunoreactivity of HIV-1 p24 and gp120 following the lysis of infected HeLa cells.

In the immunoplaque assay, double staining is used to measure expression levels of the HIV-1 antigen and the ALVAC vector, respectively, in order to demonstrate a consistent ratio between antigen and vector expression levels [15]. In CEF, antigen expression levels are however 100%. In contrast, only 70% of infected HeLa cells appear to express HIV-1 antigens, suggesting that only limited amounts of these are produced during early gene expression in HeLa cells, which are nonpermissive for ALVAC replication [14, 15]. This finding suggests that these tests are more important for establishing the consistency of potency of vaccine batches; they would not by themselves provide any certain predictive measure ofvaccine efficacy in humans. It is evident that the level of expression of heterologous antigen is dependent on the type of cell infected, and could be quite variable. In addition, it remains unclear as to how important correct post-translational modification, such as glycosylation, is for antigen presentation and immunogenicity.

Other viruses also show promise for the development of recombinant viral vector vaccines; these include human and animal adenoviruses, and certain alphaviruses.

Foreign DNA may be readily incorporated into adenoviruses by replacement of the viral early E1 sequences with a heterologous gene construct [16, 17]. The resulting recombinant adenoviral vector can be propagated in "special" E1-complementing cell lines, but is otherwise replication-deficient in human cells. Nevertheless, as with recombinant poxviral vectors, the heterologous gene is expressed in human cells to provide measurable amounts of the intended antigen. Recombinant adenoviral vectors have been widely used in the gene therapy field (for monogenic disorders, malignancies and cardiovascular disease) [16, 17], but are now increasingly being developed as prophylactic vaccines, particularly against HIV-1. For example, recombinant adenovirus type 5 vectors expressing consensus genes encoding HIV-1 clade B gag or trivalent gag/pol/nef are being tested as HIV-1 vaccines in clinical trials to assess safety and efficacy [95, 99]. As in the case of recombinant poxviral vectors, levels of antigen expression may be measured in vitro following infection of suitable cell lines. Again, it is not possible to predict immunogenicity/efficacy in vivo simply by reference to antigen expression levels in vitro.

Alphavirus "replicon" vectors based on the RNA viruses Semliki Forest virus (SFV), Sindbis virus (SINV) and Venezuelan Equine Encephalitis virus (VEE), are being proposed as potential preventative vaccines [15, 18-21], especially against HIV-1 and influenza viruses, but are not as advanced in their development as recombinant poxviral or adenoviral vector vaccines. They are produced by replacing the alphaviral structural genes with a heterologous gene insert, which renders the recombinant replicon vectors propagation-incompetent. The RNA replicons may be packaged into virus-like particles, or alternatively they can be converted into their corresponding functional DNA counterparts that directly transcribe the RNA replicons in situ. The alphavirus DNA replicon is considered to be essentially similar in physical properties to "conventional" plasmid DNA vaccines. However, in contrast to nonreplicating plasmid DNA, translation of the replicon RNA produces the alphavirus replicase complex, which catalyzes cytoplasmic self-amplification of the replicon RNA and, advantageously, leads to high-level synthesis of the heterologous antigen [18]. Such replicon RNA self-amplification causes cell death within a few days and is thus self-limiting. Following vaccination with DNA replicons, it is therefore expected that the expression of an antigen-coding gene would be at high level, but transient in duration [18]. This compares with plasmid DNA vaccines where plasmid DNA persists for months and expression of the antigen-coding gene is low level.

Another factor in the equation of how efficacious the vaccine will eventually be is whether the subjects to be immunized have pre-existing immunity to the virus vector. However, it may be assumed that humans will not have pre-existing immunity to canarypox virus on which ALVAC is based. Even those people immunized against smallpox with a vaccinia vaccine between the 1950s and 1970s will have little immunity left against vaccinia or its attenuated derivatives, such as MVA. Indeed, it has been shown recently that priming and amplification of immune responses specific for heterologous antigens expressed following administration of recombinant MVA can be achieved. Nevertheless, the primary use of recombinant MVA or ALVAC vaccines will, in most cases, trigger some immunity against their secondary usage, and thus potentially reduce their efficacy when subsequently used in the same subjects. The outlook for recombinant adenoviral vector vaccines is similar; there is often some pre-existing immunity to human adenoviruses in the population, as these are the cause of common respiratory infections. The use of less-prevalent human adenoviruses - for example, Ad6, Ad35, Ad11 or Ad24 [95, 100, 101], or more exotic animal adenoviruses [22] - as bases for the development of recombinant adenoviral vector vaccines may circumvent the potential problem of pre-existing immunity. Again, the primary use of such recombinant adenoviral vector vaccines may preclude any further secondary use with the same vaccine, or another vaccine based upon the same adenovirus, because of immunity to the adenoviral vector "backbone".

In summary, the predictive power of antigen-expression tests in vitro remains limited and uncertain. It is evident that it is difficult to mimic the in-vivo situation at the site of immunization within a single cultured cell line. As the exact phenotype of cells infected by the viral vector is not easy to establish, the antigen-expression tests in a particular cell line in vitro can only establish the level of antigen expression there; expression at the immunization site, while appearing probable, is uncertain both in the level and length of time over which expression may occur. The possibility of pre-existing immunity to the viral vector itself can add to the uncertainty of the efficacy of the vaccination. Therefore, measurements of antigen expression in vitro are only able to serve as a guide to potential expression in vivo. They do not by themselves reliably indicate the immunogenicity or, ultimately, the efficacy of the vaccine. Other tests are required to correlate with efficacy; the results of these may relate back to antigen expression in vitro and the combination of results may be of more practical value than those of the individual tests alone.

158 | 6 Cell Lines and Primary Tissues for In-Vitro Evaluation of Vaccine Efficacy 6.3

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