Marion E. Perkus

Host-range restriction of vaccinia virus E3L-specific deletion mutants

The vaccinia virus (VV) E3L gene product functions as a dsRNA binding protein that is involved in conferring an interferon-resistant phenotype upon the virus. Studies with a vaccinia virus (VV) E3L- deletion mutant (vP1080) have also demonstrated that the E3L gene product is critical for productive replication on certain cell substrates. While E3L was found to be nonessential for replication in chick embryo fibroblasts (CEFs), virus specifically deleted of E3L was found to be replication deficient in Vero, HeLa, and murine L929 cells. Further, the temporal block in replication appears to differ in these cell systems, as evidenced by the observed timing of protein synthesis inhibition. In Vero cells infected with the VV E3L- mutant, there was no detectable protein synthesis after 2 hr post-infection, whereas in L929 cells normal protein patterns were observed even at late times post-infection. Expression of a heterologous dsRNA binding protein, the reovirus σ3 protein, by the E3L- mutant virus restored near wild-type growth characteristics, suggesting the critical nature for regulating dsRNA levels in VV-infected cells.

POXVIRUS-BASED VACCINE CANDIDATES FOR CANCER, AIDS, AND OTHER INFECTIOUS DISEASES

Over the past 12 years, the poxvirus vector technology has provided scientists with valuable reagents to achieve high‐level expression of proteins, to address questions of structure‐function relationship of specific polypeptides, to investigate the immunobiology of specific pathogens, and to develop recombinant vaccine candidates. It is this last role that has drawn enthusiasm from the medical community because of the potential this technology has to provide novel approaches for addressing urgent needs in human and veterinary medicine. From one perspective, the safety issues surrounding the use of vaccinia‐based vaccine candidates have been addressed with the development of the NYVAC and ALVAC vectors. Evaluation of these novel poxvirus vectors are in progress to determine their potential impact on cancer and infectious disease. J. Leukoc. Biol. 58: 1–13; 1995.

Vaccinia virus as an expression vector.

Publisher Summary The chapter describes vaccinia virus as an expression vector. It describes the methodology for using vaccinia virus to express foreign genes. These recombinant vaccinia viruses can be used for several purposes:(1)understanding the genetic regulation involved in the expression of endogenous vaccinia functions, (2) determining the fate of foreign gene products in a background free of native influences, (3) producing biologically active molecules, (4) elucidating the roles of pertinent antigens in eliciting defined immunological responses, (5) producing live recombinant vaccines, and (6) potentially using gene replacement therapy. The chapter discusses the biology of poxviruses stating that they comprise a large group of complex animal viruses, whose genetic information is contained within a double-standed DNA molecule and whose replication is confined to the cytoplasm of the infected cell. The chapter describes the principle of the method regarding the general protocol for the insertion of foreign genes into vaccinia virus. Vaccinia displays a number of characteristics, which make it an ideal expression vector. Some of these are as follows:(1) the ability to incorporate large amounts of exogenous DNA, (2) the faithful transcription of the exogenous genes such that the RNAs are translated into protein, which resemble the native product in structure, function, and localization, (3) the cytoplasmic site of vaccinia replication, allowing gene expression to proceed without interference by the host genome, and (4) the potentiality as a live recombinant vaccine as a target against human and veterinary pathogens. The future research efforts will focus on the manipulation of the vaccinia genome with respect to defining the genetic parameters, which control the expression of both endogenous and exogenous genes.

Antibody-dependent cellular cytotoxicity is directed against both the gp120 and gp41 envelope proteins of HIV.

To define the target antigens for antibody-dependent cellular cytotoxicity (ADCC), assays were performed using affinity-purified human immunoglobulin (Ig) or polyclonal rabbit sera directed against specific proteins of HIV. ADCC was not found using affinity-purified anti-core (p25) human Ig or sera obtained from rabbits hyper-immunized with recombinant p25. However, when affinity-purified human Ig or rabbit antisera specific for the envelope glycoproteins, gp120 or gp41, were used in ADCC assays, killing of HIV-infected cells was observed. These results indicate that antibodies in the infected individual that mediate ADCC are directed against both the gp120 and gp41 HIV envelope proteins and not against the viral core protein.

A Modern Approach to Live Vaccines: Recombinant Poxviruses

A technique for preparing live recombinant vaccines is described. The technique is a blend of old and new technologies. Vaccinia virus, used for almost two hundred years in the immunoprophylaxis of smallpox, has been engineered by recombinant DNA technologies to express foreign genetic information derived from heterologous pathogens. This recombinant live vaccine virus has been shown to elicit important immunological responses to these foreign antigens on inoculation of the recombinant virus into animals. Significantly, a number of studies have shown that vaccination of laboratory animals with these recombinant viruses results in protecting these animals against disease on subsequent challenge with the heterologous infectious agent. Vaccinia virus recombinants expressing the influenza virus hemagglutinin, the herpes simplex virus glycoprotein D, the hepatitis B virus surface antigen, the rabies virus glycoprotein, and a malarial parasite antigen are described and the biological properties of these recombinant viruses as live immunogens are detailed. A brief description of the problems and future prospects is included.