A Pilot Study of the Humoral Response Against the AntiSense Protein (ASP) in HIV-1-Infected Patients.

The existence of an antisense Open Reading Frame (ORF) that encodes a putative AntiSense Protein (ASP) on the proviral genome of Human Immunodeficiency Virus type 1 (HIV-1) was a source of debate for 30 years.

During the last years, some progresses have been made to characterize the cellular immune response against ASP in HIV-1 seropositive patients. However, no tools were available for the detection of antibodies to ASP in the plasma of HIV-1-infected patients during the natural course of the infection.

The aim of our study was to develop a Luciferase Immuno-Precipitation System (LIPS) to monitor the quantitative detection of ASP-specific antibodies in the plasma of HIV-1-infected patients [antiretroviral therapy (ART) naive-patients, patients under ART and HIV-1 controllers], patients who discontinued antiretroviral drugs (ARV). We further used this approach to delineate the epitopes of ASP targeted by antibodies.

Antibodies directed against ASP were detected in 3 out of 19 patients who discontinued ARV (15%) and in 1 out of 10 ART-naive patients (10%), but were neither detected in HIV-1 infected patients under ART nor in HIV-1 controllers. Individual variations in levels of ASP-specific antibodies were detected overtime.

Both the conserved prolin-rich motif and the core 60-189 region of ASP were found to be essential for antibody recognition in the four patients tested positive for anti-ASP antibodies, who were all untreated at the time of sampling.

Moreover, for two of these patients, increased levels of ASP-specific antibodies were observed concomitantly to viremia declines. Overall, our method may represent a useful tool to detect a humoral response to ASP in HIV-1-infected patients, which allowed us to confirm the expression of ASP during the course of HIV-1 infection. Further studies will be needed to fully characterize the humoral response to ASP in HIV-1-infected patients.


Comparison of two Immunoassays for Concurrent Detection of HCV Antigen and Antibodies among HIV/HCV Co-infected Patients in Dried Serum/Plasma Spots.

Hepatitis C virus (HCV) antigen/antibody (Ag/Ab) assays offer the benefit of reducing the window period compared to assays that detect only HCV-Ab. In this study the performance of the Murex Ag/Ab (Murex, Abbott) and Monolisa Ag/Ab Ultra (Monolisa, Bio-Rad) ELISAs was compared for the use of filter dried serum/plasma spots (DS/PS) with a focus on the sensitivity and the percentage of correct positive test results.

Correct positive ELISA results were assumed for samples that subsequently tested positive for HCV RNA by RT-qPCR, or RNA negative samples that tested positive in a Western blot (confirmed ELISA results).

Sensitivity was evaluated from DS/PS eluates using HCV seroconversion panels [plasma samples of subtypes-(St) 1a, 2b)] and longitudinal HCV antibody-positive serum panels (St 1b, 2b, 3a, and 4d). The proportion of correct positive test results was evaluated using 1102 newly-diagnosed HIV-positive clinical dried serum spots DSS eluates for screening of potential HCV co-infection.

For the plasma HCV seroconversion samples, which were used as a reference for DSS eluates, the Murex became reactive earlier for antigen-positive bleeds. However, for the HCV antibody-positive eluates and dilutions thereof, the Monolisa demonstrated a superior sensitivity.

Of the clinical DSS 22.8% (28/123) of samples reactive in the Murex were negative in a subsequent RT-qPCR and Western blot, while only 1.9% (2/105) of the samples reactive in the Monolisa were negative in these confirmatory assays. Our results indicate that the Monolisa provides fewer false positive results for HCV detection in DSS, whereas for undiluted plasma or serum samples, the Murex can serve as an additional diagnostic tool to narrow the window period.

CRISPR/Cas systems for editing genes in HIV treatment Research

What are the riscks in using gene therapy are we close to approval of such treatments?

The main concern is that gene therapy might create a Frankenstein cell, they would hit different genes rather than the intended goal, so it is great that this did not occur.China appears to be moving fast on such research and may get remedies approved earlier than the United States, June explained. He’s financial ties to some gene therapy companies and is leading another study testing CRISPR to fight cancer in the united states. Three patients have been treated up to now and some results are expected at the end of this season.

What is CRISPR/Cas9 system ?

CRISPR -clustered regularly interspaced short palindromic repeats is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences have been derived from DNA fragments of bacteriophages that had formerly infected the prokaryote.CRISPR works in coordination with CRISPR associated (CAS) genes, enabling prokaryotic adaptive immune systems to comprehend, react to, and remove foreign DNA. CRISPR/Cas chemical editing is called a molecular scissors since it essentially cuts the foreign DNA (plasmid and bacteriophage) from the prokaryotic genome. Analogies of CRISPR/Cas gene editing in prokaryotes are created to RNA interference or receptor found in eukaryotic organisms

CRISPR/Cas gene editing mode of action

Could CRISPR/Cas system be used for inactivation of  HIV infection?

Scientists have made RNA guides to guide Cas9 to cleave unique areas of HIV viral DNA comprising essential genes along with the long terminal repeat.3 CRISPR/Cas9 gene editing led to significant reduction of HIV viral generation and disease in various research mobile versions, such as human pluripotent stem cells, primary CD4+ T cells, and CD4+ T cell lines. Theoretically, the removal of HIV viral DNA or enzymes necessary for viral expression in infected cells need to have the ability to inactivate or eliminate HIV disease.

Regrettably, researchers also revealed cells developed CRISPR/Cas9 resistance mutations in certain experiments. All these mutations clustered in the viral website once the Cas9 was led . Cas9 wasn’t able to cleave the targeted website and thus the CRISPR/CAS9 strikes were unsuccessful.HIV, like other viruses, was known to possess a well-developed capability to evolve immunity to other assaulting mechanisms like the human immune system and anti inflammatory drugs.

CRISPR/CAS9  HIV mechanism
CRISPR/CAS9 mode of action in HIV infected cells

What is the most successful strategy for gene therapy of HIV, so far?

Combating HIV-1 disease, CRISPR/Cas9 was used to ablate chemokine receptor type 5 (CCR5) expression. CCR5 is a co-receptor and necessary for specific HIV types to get entrance into T-cells and lead to HIV disease. The mechanics of CCR5-ZFN relies on genetically engineered alterations of CCR5 that make CCR5 non-functional and tissues resistant to HIV infection. What is yet to be found is whether CCR5-ZFN/SB-728-T could replicate itself to further resistant cells and may restore immune cell functioning in infected hosts by preventing HIV entry into cells.