HIV-1 cDNA in Semen

Episomal 2-long terminal repeat (LTR) HIV-1 cDNA, a by-product of HIV-1 infection, is used in clinical trials as a marker for ongoing viral replication. It would be useful to employ 2-LTR cDNA to monitor cryptic HIV 1 infection in the genitaltract of men on antiretroviral therapy (ART) to predict the evolution of sexually transmissible drug-resistant HIV-1, but studies thus far have failed to detect this marker in semen.

The global AIDS epidemic is primarily attributed to the sexualtransmission of Human Immunodeficiency Virustype-1 (HIV-1), a retrovirus that infects CD4+ T cells and macrophages and causes severe immunosuppression in most untreated infected individuals. In HIV-1-infected men, sexual transmission rates are high during acute infection and late-stage disease when HIV-1 viral loads are elevated in both semen and peripheral blood [1, 2].

Materials And Methods Semen processing

The semen samples used in this study were archived from previously

published studies [1, 6]. Positive control samples used for validation of 2-LTR cDNA detection/quantification were from ART-naïve men with evidence of seminal HIV-1 viremia (Group I). HIV-1 proviral DNA in the cellular fraction had been assessed by quantitative PCR; infectious HIV-1 was detected in seminal plasma and cellular fractions by p24 assay following up to 21 days of coculture with phytohaemagglutinin A (PHA)-stimulated peripheral blood mononuclear cells (PBMC) [1].

The PCR productswere also analyzedby agarose gel electrophoresis, Southernblot and DNA sequencing. Fifteen microliters of PCR product were separated on a 1% agarose gel and transferred to a Hybond N+ membrane(Schleicher & Schuell, Keene, NH); Southern blot was performed using the 29-base biotinylated probe. After a high-stringency wash, streptavidin-HRP conjugate and its substrate in Luminol/enhancer solution (Pierce, Rockford, IL) were added to the membrane. The image was acquired with Fluorchem SP (Alpha Innotech,San Leandro, CA). Direct sequencing of the PCR product was infected cells in semen is restricted by small sample size, steps must be taken to maximize HIV-1 DNA extraction. In the only other published study to investigate 2-LTR cDNA in semen, Nunnari et al.

All of the 2-LTR cDNA+ semen samples contained HIV-1 proviral DNA, and all but two were positive for HIV-1 RNA or infectious virions. Five out of 7 of the 2-LTR cDNA+ semen samples were also positivefor seminal CMV DNA, but this association was not statistically significant because CMV infection was common overall (4/8 men in Group I and 9/22 men in Group II were positive for seminal CMV).

Two men on dual nucleoside analog treatment had 2-LTR cDNA in semen;one of these individuals failedsubsequent indinavir therapyand continued to have high levels of HIV-1 RNA and 2-LTR cDNA in semen, although peripheral viralload was undetectable. These data provide further evidence that HIV-1 replication can occur in the genital tract of men on ART, potentially leading to the evolution of drug resistance mutations in semen that cannot be monitored in peripheral blood. On the other hand, none of 40 post-indinavir semen samples from men controlling HIV-1 RNA levels in blood and semen was positive for 2-LTR cDNA, suggesting that cryptic HIV-1 replication in the genital tract (without the appearance of HIV-1 RNA in blood or semen)may be uncommon in men on effective ART [6].                                                                                                                                                                                                                                                                             

Results

Many semen sampleswith indicators of HIV-1 infection were negative in the 2- LTR cDNA assay. Of the four negative GroupI samples, all were from ART-naïve men with seminalHIV-1 proviral DNA (inclusion criteriafor Group I); one was culture-positive for HIV-1, and 2 were CMV DNA+. Of the 20 2-LTR cDNA- negative Group II baseline samples, one was leukocytospermic, 4 had >100 copies of HIV-1 proviralDNA, 5 contained >1,000 copiesof cell-free HIV-1 RNA and 6 were CMV DNA-positive. Of the 40 2-LTR cDNA-negative post-indinavir semen samples, four were leukocytospermic, 12 had proviral HIV-1 DNA, and 5 were from men with seminal CMV DNA.

Discussion

The highest copy numbers of 2-LTR cDNA in this study were detected in semen of a man failing indinavir therapy who later developed unique PI resistance mutations in semen. Data from this study suggest that seminal 2-LTR cDNA may providea useful markerto predict the evolution of sexually transmissible

Conclusion

This study, which shows that HIV-1 2-LTR c-DNA is detectable in semen cells, provides evidence that HIV-1 infection occurs in the genital tract.

References

Performed by the Dana-Farber/Harvard Cancer Center core facility.