Enzo Paoletti

Recombinant fowlpox virus inducing protective immunity in non-avian species

The natural host of fowlpox virus is limited to avian species. When inoculated into non-avian tissue culture cells, however, fowlpox virus can initiate an abortive infection. A fowlpox virus was engineered to express rabies virus glycoprotein. On inoculation of the recombinant virus into either avian (permissive) or non-avian (non-permissive) cells, the rabies glycoprotein was expressed as a membrane-associated antigen. Inoculation of the fowlpox virus recombinant into six different species of mammal resulted in specific immune responses to both fowlpox antigens and to rabies glycoprotein. In mice, cats and dogs the immune response was sufficient to protect against a live rabies virus challenge. The results demonstrate the utility of a fowlpox virus vector in immunizing non-avian species against rabies in the absence of productive viral replication of the fowlpox vector.

Fowlpox virus as a vector in non-avian species.

Examination of the members of the Poxvirus family reveals a large and diverse group with members infecting almost every animal species. To a large extent, members of the individual genera have a broad host range and infect a number of animal species, although, as Baxby has pointed out, successful experimental inoculation of a species does not necessarily mean that this species provides a natural host for that virus. Avipox viruses, together with swinepox virus occupy a somewhat unique position in possessing a restricted host range. We have taken advantage of this restricted host range in fowlpox virus to engineer recombinant vector viruses for use in the poultry industry and in vaccination of non-avian species.

Insertion and deletion mutants of vaccinia virus

Thirteen viable insertion mutants of vaccinia virus have been constructed. These mutants, containing coding sequences of the herpes simplex virus thymidine kinase (HSV-TK) gene, were generated by marker transfer via in vivo recombination. The mutants were identified using a replica filter plating technique by in situ hybridization using 32P-nick translated HSV-TK sequences and obtained as pure cultures by repeated plaque purification. Some of these insertion mutants were in turn used as substrates to generate viable deletion mutants of vaccinia virus in the presence of 5-bromodeoxyuridine. An example of this approach resulting in a vaccinia virus deleted of approximately 1.5 kb of nonessential DNA is presented. Furthermore, the analysis of spontaneously occurring viable deletion mutants of vaccinia lacking approximately 21.4 kb of nonessential DNA is described.

Vaccinia: Virus, Vector, Vaccine

Publisher Summary This chapter discusses the genetic characterization of vaccinia, a general protocol for the insertion of foreign genes into vaccinia virus, and analysis of the recombinant viruses. The vaccinia virus can infect a variety of animals and is able to replicate in a variety of tissue culture cells. When experimenting with this virus, two unique viral features must be taken into account: (1) the virus replicates independently within the cytoplasm of the infected cell, and (2) the naked DNA of vaccinia is not infectious, unlike the DNA of many other animal viruses, such as herpesviruses, adenoviruses, and papovaviruses. These features, combined with the large DNA size, disallow it from being used as a cloning vector via the “cut and paste” approach commonly used for other viral genomes and plasmids. Instead, the ability to introduce genetic elements into the vaccinia genome is accomplished by marker rescue techniques. The recombinants of vaccinia virus can be used as expression vectors with biological applications ranging from cloning and expression vehicles to live vaccines directed against infectious diseases of both human and veterinary concern.

The phosphorylation of the integral membrane (M1) protein of influenza virus

The phosphorylation of the internal and integral membrane (M1) protein of influenza virus was studied. Four points can be made based on the data: (1) The M1 contains at least two moles of phosphate per mole of M1. (2) Phosphorylation of M1 is conserved between influenza A, B and C viruses. Other characteristics of the M1 are also conserved, such as solubility in organic solvent, heterogeneity and ability to partition into lipid vesicles. (3) M1 is phosphorylated in cells infected with a vaccinia recombinant (vP273) containing only the gene of M1, either as a result of a vaccinia virus associated kinase or a cellular one. (4) The phosphate is located within or in close proximity to the major stretch of neutral and hydrophobic amino acids found in M1, as determined by analyzing cyanogen bromide fragments.

Molecular complexity of vaccinia DNA and the presence of reiterated sequences in the genome

Abstract Reassociation rate measurements suggest that the molecular weight of the DNA of vaccinia virus is 1.2 × 10 8 , when polyoma virus DNA is used as a standard of comparison. In addition, a search for reiterated sequences revealed that a small portion of the genome (0.6%) occurs as multiple copies.

Genetically engineered poxviruses: a novel approach to the construction of live vaccines.

Gene splicing techniques have been used to modify the smallpox vaccine virus thus providing a generic approach for the construction of live vaccines directed against a variety of heterologous infectious disease agents. The technique involves translocating a particular gene from an infectious agent into the genetic material of the smallpox vaccine virus. This unique foreign gene, selected because it contains the information essential for the synthesis of an antigen important in immunity to that particular infectious disease agent, is now expressed under the regulation of the engineered smallpox vaccine virus. On immunization with this live recombinant vaccine, the body is fooled into thinking that it was infected by the foreign infectious disease agent and mounts a defensive attack resulting in immunity to that particular infectious agent. Three examples of this approach are provided. Thus, smallpox vaccine viruses were engineered to express genes encoding the hepatitis B virus surface antigen (HBsAg), the herpes simplex virus glycoprotein D (HSV-gD) and the haemagglutinin (HA) from influenza virus. These foreign gene products when synthesized in vitro under vaccinia virus regulation were shown to be antigenic by a variety of serological tests. When these recombinant vaccinia viruses were inoculated into laboratory animals, the heterologous gene products elicited the production of specific antibodies thus demonstrating that they were immunogenic. Serum neutralizing antibodies were demonstrated to be present for both influenza and herpes simplex viruses. Additional studies in mice showed that a recombinant smallpox vaccine virus expressing a gene from herpes simplex virus effectively protected the mice when subsequently challenged with what would normally be lethal doses of infectious herpes simplex virus.

The role of ATP in the biogenesis of vaccinia virus mRNA in vitro

Abstract During in vitro RNA synthesis vaccinia virus rapidly depletes ATP but not GTP, CTP, or UTP. This reduction in ATP concentration results in a decreased rate of RNA synthesis. The RNA transcripts made when ATP becomes rate-limiting are large in size, 20 to 30 S, compared with the 8 to 12 S RNA transcripts synthesized under high ATP concentration. Furthermore, these large RNA transcripts are not significantly polyadenylated and remain virion-associated. When one supplements the RNA polymerase reaction with additional ATP the following observations are made: (a) The rate of RNA synthesis remains constant; (b) the RNA synthesized is of the appropriate monocistronic size, 8 to 12 S; (c) the RNA is polyadenylated efficiently; and (d) the RNA is released from the virus. Some of these effects are mimicked by dATP, but neither the αβ- nor the β,γ-phosphonic acid analogs of ATP will replace ATP in the supplementation scheme. Selection of appropriate ATP concentrations in the in vitro RNA polymerase reaction allows exclusive synthesis of high-molecular-weight RNA.

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.