Elucidating and Minimizing the Loss by Recombinant Vaccinia Virus of Human Immunodeficiency Virus Gene Expression Resulting from Spontaneous Mutations and Positive Selection

Abstract

While characterizing modified vaccinia virus recombinants (rMVAs) containing human immunodeficiency virus env and gag-pol genes, we detected nonexpressing mutants by immunostaining individual plaques. In many cases, the numbers of mutants increased during successive passages, indicating strong selection pressure. This phenomenon provided an opportunity to investigate the formation of spontaneous mutations in vaccinia virus, which encodes its own cytoplasmic replication system, and a challenge to reduce the occurrence of mutations for vaccine production. Analysis of virus from individual plaques indicated that loss of expression was due to frameshift mutations, mostly by addition or deletion of a single nucleotide in runs of four to six Gs or Cs, and large deletions that included MVA DNA flanking the recombinant gene. Interruption of the runs of Gs and Cs by silent codon alterations and moving the recombinant gene to a site between essential, highly conserved MVA genes eliminated or reduced frameshifts and viable deletion mutants, respectively. The rapidity at which nonexpressing mutants accumulated depended on the individual env and gag-pol genes and their suppressive effects on virus replication. Both the extracellular and transmembrane domains contributed to the selection of nonexpressing Env mutants. Stability of an unstable Env was improved by swapping external or transmembrane domains with a more stable Env. Most dramatically, removal of the transmembrane and cytoplasmic domains stabilized even the most highly unstable Env. Understanding the causes of instability and taking preemptive actions will facilitate the development of rMVA and other poxviruses as human and veterinary recombinant vaccines.

Genomic Analysis of the Vaccinia Virus Strain Variants Found in Dryvax Vaccine

“…It is important to note that many of the insertions and deletions associated with 1 to 3 nt of identity occur at sites of repeated sequence (e.g., a T from TTT or an AT from ATAT) and thus tend to skew these statistics, even when we discount deletions occurring in longer patches of homopolymeric repeated sequence as being probable artifacts of the sequencing technology. A similar pattern of Streisinger frameshifting within short homopolymer repeats has been previously noted in recombinant VACV MVA strains carrying cloned HIV genes (38). The number of events declines as the length of the duplication increases, possibly due to the greater instability of longer repeats and thus their deletion from the virus pool.…”

A Vaccine Based on the Rhesus Cytomegalovirus UL128 Complex Induces Broadly Neutralizing Antibodies in Rhesus Macaques

“…The individual gene expression cassettes were inserted into separate insertion sites in opposite transcription orientations to allow comparable transgene expression while reducing the risk of homologous recombination between the promoter elements (Fig. 1B) (48). The integrity of the cloned MVA genome, as well as markerless insertion of the RhCMV genes, was confirmed by restriction fragment analysis, PCR, and sequencing (data not shown).…”

Mucosal Immunization of Lactating Female Rhesus Monkeys with a Transmitted/Founder HIV-1 Envelope Induces Strong Env-Specific IgA Antibody Responses in Breast Milk

“…Plasmid p2614 contains a chemically synthesized 3,853-nucleotide SacI-ApaI fragment containing the following: (i) a copy of the gene encoding the C.1086 Env protein in which the nucleotide sequence was modified where possible by silent codon alterations to disrupt runs of 4 to 5 consecutive G's or C's, as well as potential early termination signals (TTTTTNT); (ii) a synthetic poxvirus early/late promoter comprising the early promoter from the cowpox virus CrmA gene (29,30) in tandem with the late promoter of the vaccinia virus p4b gene (31) to direct transcription of the gp140 gene; (iii) an early termination signal immediately after the introduced stop codon in the gp140 gene; (iv) a marker cassette containing the K1L and GUS genes from p2588 flanked by 200-bp direct repeats of the 3= end of the K1L gene to permit transient marker stabilization and selection of the gene insertion into the MVA genome; and (v) flanking regions to direct insertion of the gp140 gene and marker cassette into the intergenic region between the two essential viral genes I8R and G1L. The removal of runs of G's and C's was done to minimize spontaneous frameshift mutations within the gp140 sequence, and the insertion of the gp140 gene between two essential MVA genes was done to minimize the potential loss of the inserted gene through spontaneous gene deletions in the viral genome, as described previously (32). After transfection of insertion plasmid DNA into cells infected with A681 virus, recombinant viruses capable of replication in RK13 cells were selected (containing the gp140 gene, the GUS gene, and the K1L gene) and then passaged in BHK21 cells, without selec-tive pressure for the K1L gene.…”