Expression of all six human Torque teno virus (TTV) proteins in bacteria and in insect cells, and analysis of their IgG responses

Abstract

Torque teno virus (TTV) is a non-enveloped human virus with a circular ( approximately 3800 nt) ssDNA genome. TTV transcription results in three viral mRNAs and six proteins, the function or antigenicity of which are unknown. The six open reading frames of TTV genotype 6 were expressed in bacteria and insect cells. Expression of the ORF1/1-encoded protein was inefficient, while expression of the others was successful, with ORF1 and ORF1/2 as arginine-rich region depleted. All six recombinant TTV proteins were antigenic. Of healthy adults, 11/25 (44%) showed strong IgG reactivity with one or more proteins. Four subjects, two of whom were genotype-6-DNA positive, were followed. One of the latter showed concurrently a strong IgG response against the ORF1 protein. The other showed appearance of IgG against the ORF2 protein concomitantly with resolution of the genotype-6 viremia. The genotype-6 sequences remained unaltered for years, suggesting that some mechanisms other than amino acid substitutions play a role in TTV immune evasion.

Antigenic diversity and seroprevalences of Torque teno viruses in children and adults by ORF2-based immunoassays

“…Furthermore, as TTV classification is based on the ORF1 sequences, our initial attempts to classify all of our TTV sequences and match them with the ORF2 antibody data were concluded to be futile. In all, our TTV DNA prevalence among seroconverted and seroreverted children is in line with those reported in previous studies, showing TTVs to circulate commonly in the general population irrespective of the antiviral antibodies (Handa et al, 2000;Kakkola et al, 2008;Ott et al, 2000) and highlighting the remarkable ability of these viruses to evade immunity.…”

“…The ORF1 protein product of the largest coding sequence, being the putative capsid protein of TTV, would appear to be a good candidate antigen for serological assays. However, in our previous studies, ORF1 expression did not yield a full-length protein, probably due to a low translation level and degradation (Kakkola et al, 2002(Kakkola et al, , 2008Qiu et al, 2005). Although partial ORF1 expression was successful, purification under native conditions of these poorly soluble proteins turned out to be difficult (our unpublished data).…”

“…100 %) with the C-terminal half of the ORF1 protein of a genotype 1 virus, whereas Handa et al (2000), using the N-terminal half of the ORF1 protein as antigen, reported a seroprevalence of 38 %. On the other hand, measuring IgG with an ORF1 genotype 6 (HEL32)-derived antigen, Kakkola et al (2008) observed much lower seroprevalences with both C-terminal (14 %) and N-terminal (0 %) protein regions, pointing to epidemiological differences between TTV genotypes. Nearly onefifth of the same serum samples exhibited seroreactivities towards the ORF2 protein of a single TTV strain, indicating the absence of antigenic cross-reactivity.…”

“…On the other hand, the ORF2 proteins could be strongly expressed in insect cells and easily purified. According to immunoblot studies by Kakkola et al (2008), the use of the ORF2 antigen as opposed to the other five TTV proteins enables serodiagnosis of the highest sensitivity, indicating that the ORF2 protein is very antigenic.…”

“…Thus far, only a few studies have addressed TTV antibodies, by employing methods such as immunoblotting, immunoprecipitation and immunocapture PCR (Handa et al, 2000;Kakkola et al, 2008;Ott et al, 2000;Tsuda et al, 1999Tsuda et al, , 2001). Because of small-sized study groups or methodological limitations, the seroprevalence assessments call for reappraisal.…”