ABSTRACT
An effective vaccine for AIDS may require development of novel vectors capable of eliciting long-lasting immune responses. Here we report the development and use of replication-competent and replication-defective strains of recombinant herpes simplex virus (HSV) that express envelope and Nef antigens of simian immunodeficiency virus (SIV). The HSV recombinants induced antienvelope antibody responses that persisted at relatively stable levels for months after the last administration. Two of seven rhesus monkeys vaccinated with recombinant HSV were solidly protected, and another showed a sustained reduction in viral load following rectal challenge with pathogenic SIVmac239 at 22 weeks following the last vaccine administration. HSV vectors thus show great promise for being able to elicit persistent immune responses and to provide durable protection against AIDS.
Subject(s)
SAIDS Vaccines/immunology , Simian Acquired Immunodeficiency Syndrome/prevention & control , Simian Immunodeficiency Virus/immunology , Simplexvirus/immunology , Vaccines, Synthetic/immunology , Animals , Antibodies, Viral/biosynthesis , Injections, Intravenous , Macaca mulatta , SAIDS Vaccines/biosynthesis , SAIDS Vaccines/genetics , Simian Acquired Immunodeficiency Syndrome/immunology , Simplexvirus/genetics , Simplexvirus/metabolism , Vaccines, Attenuated/biosynthesis , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Vaccines, Synthetic/biosynthesis , Vaccines, Synthetic/genetics , Viral Envelope Proteins/immunology , Viral Envelope Proteins/metabolism , Viral Load , Virus ReplicationABSTRACT
An efficient method for generating full-length DNA sequences from apparently unsuccessful polymerase chain reactions (PCR) has been developed. In cases where nonspecific background interferes with detection of the PCR product, a second amplification is performed using a nested set of primers. The internal fragment of DNA amplified in this reaction is then blotted to a membrane and used to hybrid-select the desired DNA from the initial amplification. This DNA is eluted and used as the template for a third round of PCR. The re-use of the original primers from the initial reaction enables the final PCR to generate full-length DNA. This technique was used to clone a full-length gene segment 8 from a mutant influenza A/WSN/33 (H1N1) virus after initial PCR attempts had failed.
Subject(s)
Polymerase Chain Reaction/methods , Base Sequence , DNA, Viral/genetics , Influenza A virus/genetics , Molecular Sequence Data , Nucleic Acid Hybridization , Sensitivity and SpecificityABSTRACT
Virus isolated from a persistent infection initiated in BHK cells with influenza A/WSN/33 (H1N1) produced an unusual pattern of protein synthesis in productive infections of BHK cells: The levels of NP and M1 proteins were slightly reduced compared to an infection with wild-type WSN, while the other proteins (Pb1, Pb2, Pa, HA, NS1, and NS2) were synthesized at very low or undetectable levels. In addition, a new viral protein with an approximate molecular weight of 11 kDa (Pi protein) is made (Frielle et al., Virology 138, 103-117, 1984). When viral RNA was analyzed by the Northern blot technique, a deletion was found in the NS gene segment and in NS1 mRNA; all other RNAs were full-sized. Immunoprecipitation of in vitro translation products demonstrated that the Pi protein reacts specifically with anti-NS1 serum. In addition, the Pi protein, like the NS1 of the parental wild-type virus, accumulated in the nucleus of infected cells. These results indicate that the Pi protein is a mutated form of the NS1 protein encoded by a deleted NS segment and suggest that this mutation may be involved in the expression of the persistent virus phenotype.
