Overview

Identification and Characterization of Retroviruses
A. Detection and identification of retroviruses using an ultrasensitive reverse transcriptase test and a highly efficient method for molecular cloning

We have developed an ultrasensitive test for the retroviral enzyme reverse transcriptase (RT) called PERT (Product-Enhanced Reverse Transcriptase) assay (U.S. Patent 5,807,669; EU Patent 0623176). The PERT assay permits detection of all retrovirus particles exhibiting an enzymatically active RT at a diagnostic sensitivity which is equal to that of specific viral RNA detection by PCR and has the additional advantage of not being negatively affected by sequence variability.

For the identification of candidate retroviruses positive by the PERT assay, but negative by PCR for known retroviral sequences, we have developed another highly efficient procedure. It permits amplification of the viral RU5 RNA sequence located at the 5'-end of the retroviral RNA from as little as 1000 copies. By making use of the redundancy of R at the 3'-end of the viral RNA genome, subsequent amplification of the entire retroviral genome by "long PCR" can be achieved.

We apply these methods to the following goals:
  • screening of human or animal disorders (cancer, autoimmune & other chronic diseases) for a possible involvement of exogenous or endogenous retroviruses.
  • safety of biomedical materials (blood products, cell cultures, vaccines etc.).
  • safety aspects of xenotransplantation.
  • characterization of candidate retroviruses thus identified.
  • quantification of specific retroviruses, HIV monitoring.
  • drug susceptibility testing (HIV).
Related publications:
  1. Pyra H, Böni J, and Schüpbach J. Ultrasensitive retrovirus detection by a reverse transcriptase assay based on product enhancement. Proc. Natl. Acad. Sci. USA 1994; 91:1544-1548.
  2. Böni J, Pyra H, Schüpbach J. Sensitive detection and quantification of particle-associated reverse transcriptase in plasma of HIV-1 infected individuals by the product-enhanced reverse transcriptase assay. J. Med. Virol. 1996; 49:23-28.
  3. Weissmahr RN, Schüpbach J, Böni J. Reverse transcriptase activity in chicken embryo fibroblast culture supernatants is associated with particles containing endogenous avian retrovirus EAV-0 RNA. J. Virol. 1997; 71: 3005-3012.
  4. Conrad B, Weissmahr RN, Böni J, Arcari R, Schüpbach J, Mach B. A human endogenous retroviral superantigen as candidate autoimmune gene in type 1 diabetes. Cell 1997; 90:303-313.
  5. Deichmann M, Huder JB, Kleist C, Naher H, Schüpbach J, Böni J. Detection of reverse transcriptase activity in human melanoma cell lines and identification of a murine leukemia virus contaminant. Arch. Dermatol. Res. 2005; 296:345-352.
     
B. Interactions of avian or porcine endogenous retroviruses with humans or human cells following enteral or parenteral exposure

Some medical procedures are associated with a parenteral exposure of humans to animal endogenous retroviruses. These include the endogenous avian retrovirus EAV-0, which is present in all chicken-cell-derived vaccines, and the porcine endogenous retrovirus (PERV), contained in porcine tissues considered for xenotransplantation.

We are investigating possible interactions of these two viruses with the human host in vitro and in vivo. Investigations address the following points:
  • the potential of these agents for infection of human cells.
  • the potential of these agents for expression in human cells .
  • the potential for interaction with co-expressed human retroviruses or members of other virus families.
  • induction of antibody responses.
  • the possibility that endogenous retroviral DNA may, as suggested by a mouse model, be taken up by the gastrointestinal tract, transported to various body tissues and lead to false-positive results in ultrasensitive molecular tests.
The proposed studies address important safety questions. Retroviral safety is a central public health issue for all medical procedures that involve animal products.
 
Related publications:
  1. Böni J, Stalder J, Reigel F, Schüpbach J. Detection of reverse transcriptase activity in live attenuated virus vaccines. Clin. Diagn. Virol. 1996; 5:43-53.
  2. Weissmahr RN, Schüpbach J, Böni J. Reverse transcriptase activity in chicken embryo fibroblast culture supernatants is associated with particles containing endogenous avian retrovirus EAV-0 RNA. J. Virol. 1997; 71: 3005-3012.
  3. Shah CA, Böni J, Bisset LR, Seebach JD, Schüpbach J. Ultra-sensitive and specific detection of porcine endogenous retrovirus (PERV) using a sequence-capture real-time PCR approach. J. Virol. Methods 2003; 109:209-216.
  4. Bisset LR, Boni J, Lutz H, Schupbach J. Lack of evidence for PERV expression after apoptosis-mediated horizontal gene transfer between porcine and human cells. Xenotransplantation 2007; 14:13-24.




Development and Evaluation of Virus-specific Diagnostic Tests
A. Measurement of HIV-1 p24 antigen by signal-amplification-boosted ELISA of heat-denatured plasma is a simple and inexpensive alternative to tests for viral RNA.

Assessment of HIV-1 RNA concentration is widely used for monitoring antiretroviral therapies. Tests are, however, expensive and require technically advanced equipment and highly trained personnel. Increasing availability of antiretroviral treatment in resource-poor settings calls for simple and inexpensive virus tests.

HIV-1 p24 antigen tests were frequently used before the availability of nucleic acid tests (NAT). Two simple modifications, heat-mediated destruction of test-interfering antibodies and increased sensitivity achieved by signal amplification, have shaped the p24 antigen test into a tool that rivals NAT. This improved p24 antigen test was evaluated in clinical studies in comparison with the most sensitive PCR methods available at a given time.

In a prospective study over 4 years, HIV-1 infection among 859 samples from 307 infants born to HIV-positive mothers in Switzerland was detected as sensitively by p24 antigen assay as by PCR for viral DNA or RNA: 100% sensitivity of all methods after 10 days of age; 99.2% diagnostic specificity of p24 after neutralization (RNA, 98.6%). A study conducted in Dar es Salaam (Tanzania) found 123 of 125 samples from 76 PCR-positive infants positive for p24 antigen (sensitivity = 98.7%). In 169 infected Swiss adults with a median CD4+ T cell count of 140 cells/µl followed for a median of 2.7 years, p24 at baseline correlated as well as or better than HIV-1 RNA with the ensuing CD4+ T lymphocyte decline and was independently predictive of progression to clinical AIDS (P = 0.043) and survival (P = 0.032). RNA predicted AIDS (P < 0.005), but not survival (P = 0.19). Another study of first-visit samples from 496 mostly black IVDU in the U.S. with a median CD4+ count of 518 cells/µl showed equally strong prediction of progression to clinical AIDS for p24 antigen, HIV-1 RNA, and CD4+ T lymphocyte concentrations at baseline. Treatment-associated changes in p24 and RNA levels in adults and children correlated well in three Swiss studies. The half-life of p24 antigen in the first phase of effective treatment was 1.6 ± 0.4 days (RNA, 1.7 ± 0.8). A second, slower decay phase had a half-life of 42 ± 16 days. One study suggested that a strategy involving a somewhat more frequent testing for p24 antigen permitted to detect viral failures significantly earlier than tests for HIV-1 RNA conducted at 3-months intervals, while at the same time significantly saving costs. Experience from three studies indicates that the p24 antigen test recognizes viruses of subtypes A - G and O, as well as some recombinant isolates, but leaves open the possibility that some non-B p24 antigens may be suboptimally detected.

This improved p24 antigen test provides diagnosis of pediatric HIV infection, prediction of prognosis and treatment monitoring in quality comparable to tests for HIV-1 RNA, but at much lower costs. There is no problem with sample instability and no need for cumbersome nucleic acid extraction. The test is validated for subtype B, but requires further studies for non-B subtypes.
 
Related publications:
  1. Schüpbach J, Böni J, Tomasik Z, Jendis J, Seger R, Kind C, and the Swiss Neonatal HIV Study Group. Sensitive detection and early prognostic significance of antigen p24 in heat-denatured plasma of human immunodeficiency virus type 1-infected infants. J. Infect. Dis.1994; 170:318-324.
  2. Sterling TR, Hoover DR, Astemborski J, Vlahov D, Bartlett JG, Schüpbach J. Prognostic value of heat-denatured HIV-1 p24 antigen and correlation with plasma HIV-1 viral load and CD4+ T-lymphocyte level in adults. J. Infect. Dis. 2002; 186:1181-1185.
  3. Schüpbach J. Measurement of HIV-1 p24 antigen by signal-amplification-boosted ELISA of heat-denatured plasma is a simple and inexpensive alternative to tests for viral RNA. AIDS Rev. 2002;4(2):83-92.
  4. Schupbach J. Viral RNA and p24 antigen as markers of HIV disease and antiretroviral treatment success. Int. Arch. Allergy Immunol. 2003; 132:196-209
  5. Knuchel MC, Jullu B, Shah C, Tomasik Z, Stoeckle MP, Speck RF, Nadal D, Mshinda H, Böni J, Tanner M, Schüpbach J. Adaptation of the ultrasensitive HIV-1 p24 antigen assay to dried blood spot testing. J. Acquir. Immune Defic. Syndr. 2007;44:247-253.  

B. Correlation of prospectively determined viral markers with the clinical and immunologic course in pediatric HIV-1 infection
 
Preliminary analysis of prospective study data from treatment monitoring in pediatric HIV-1 infection suggest that the course of p24 antigen concentrations in individual children may be more closely related to the course of CD4 cells than is the HIV-1 RNA (unpublished).

In this project are systematically evaluating the accumulated data of all patients enrolled in the Swiss Mothers & Children with HIV Infection cohort (MOCHIV) that were followed prospectively for HIV-1 RNA and p24 antigen. This relates to 20 children treated at the Children's University Hospital in Zurich and a total of 15 children from other centers from whom sequential viral load data over at least 2 years are available at the SNCR.

This evaluation will lead to a better understanding of the viral components most closely associated with the decline or increase of CD4 T-cell numbers and with clinical progression or recovery.


C. The viral protein load, as represented by HIV-1 p24 antigen, during interrupted (SSITT) and continuous (SMT) treatment 

Structured treatment interruption (STI) in the Swiss-Spanish Intermittent Treatment Trial (SSITT) has not resulted in the hoped-for benefits, as assessed by immunologic and virologic parameters, but remains an interesting treatment option due to reduced side effects and treatment costs. In a preliminary analysis of 14 Zurich patients of SSITT who were followed in the SSITT Plus study (H. Guenthard) we found that the overall concentrations of viral p24 antigen measured by a new ultrasensitive procedure decreased significantly during the four STI cycles (P<.0001), despite clear transient increases during the treatment-free intervals. The calculated half-life of p24 antigen was around 24 weeks. This decrease suggests that the virus replication permitted during treatment-free intervals does not suffice to compensate for the elimination of viral protein; there is also a possibility that the decrease in the viral protein load is due to boosting of antiviral effector mechanisms.

The following work is currently underway:
  • extension of these studies to all Swiss patients of SSITT in order to confirm our preliminary findings and to assess whether p24 antigen concentration correlates with viral, clinical and immunologic markers over the course of the study.
  • comparison of the half-life of p24 antigen under STI to that of the two treatment arms, PI-containing and Trizivir, in the Simplified Maintenance Treatment (SMT) study.
The study will indicate whether clearance of viral protein reservoirs is positively affected by STI and yield additional information on the relevance of p24 antigen as a marker of disease activity.




Updated: 03/2007
© 2009 - Swiss National Center for Retroviruses