The Differential Anti-HIV Effect of a New Humic Substance-Derived Preparation in Diverse Cells of the Immune System

The anti-HIV activity of a new humic substance-derived preparation has been studied in individual pools of immune cells (CD4+ T lymphocytes, macrophages, dendritic cells). Near-complete inhibition of the HIV infection (by more than 90%) was achieved by treating each of the abovementioned cell types with non-toxic concentrations of the preparation. The inhibitory effect demonstrates the possibility of preventing the depletion of a significant portion of functionally important immune cells. A comparative study of infection inhibition in individual cell pools has allowed us to reveal the differences in the preparation’s effectiveness in each of the cell populations. A R5-tropic HIV-1 infection in macrophages exhibited maximum sensitivity to the preparation: 90% and 50% inhibition of the infection were observed in the presence of concentrations as low as 1.4 and 0.35 μg/ml, respectively. A 15- and 19-fold higher concentration was required to achieve the same extent of inhibition in dendritic cells infected with the same strain. The effectiveness of the drug in CD4 + T lymphocytes is quite comparable to its effectiveness in macrophages. The drug is universally effective for both the T- and M-tropic variants of HIV-1

Validation of an automated system for aliquoting of HIV-1 Env-pseudotyped virus stocks

<div><p>The standardized assessments of HIV-specific immune responses are of main interest in the preclinical and clinical stage of HIV-1 vaccine development. In this regard, HIV-1 Env-pseudotyped viruses play a central role for the evaluation of neutralizing antibody profiles and are produced according to Good Clinical Laboratory Practice- (GCLP-) compliant manual and automated procedures. To further improve and complete the automated production cycle an automated system for aliquoting HIV-1 pseudovirus stocks has been implemented. The automation platform consists of a modified Tecan-based system including a robot platform for handling racks containing 48 cryovials, a Decapper, a tubing pump and a safety device consisting of ultrasound sensors for online liquid level detection of each individual cryovial. With the aim to aliquot the HIV-1 pseudoviruses in an automated manner under GCLP-compliant conditions a validation plan was developed where the acceptance criteria—accuracy, precision as well as the specificity and robustness—were defined and summarized. By passing the validation experiments described in this article the automated system for aliquoting has been successfully validated. This allows the standardized and operator independent distribution of small-scale and bulk amounts of HIV-1 pseudovirus stocks with a precise and reproducible outcome to support upcoming clinical vaccine trials.</p></div

Variability of the titration data at the dilution of 150,000 RLU.

<p>(A) PVO.4 first harvest automatically aliquoted, (B) PVO.4 first harvest manually aliquoted, (C) PVO.4 second harvest automatically aliquoted, (D) PVO.4 second harvest manually aliquoted. *Statistical difference ▲in comparison to <b>|</b> with <i>p</i><0.05.</p

Quality control of the precision of large-scale HIV-1 pseudovirus stocks.

<p>Neutralization titer comparison of automatically and manually aliquoted viruses by assaying five defined test reagents.</p

Titration data of the pseudovirus RHPA4259.7.

<p>Automatically aliquoted pseudovirus and the manually filled virus before the automated aliquoting procedure compared to manually filled virus directly frozen with the allowed 3-fold acceptance limit.</p

Titration data of the pseudovirus RHPA4259.7 displaying each channel from the first, middle and last rack aliquoted automatically.

<p>(A) first harvest with the defined 3-fold range between 11 and 102 and the %CV of 7.0, (B) second harvest with the defined 3-fold range between 4 and 33 and the %CV of 23.5.</p

Automated system for aliquoting and the associated components.

<p>(A) Complete view of the system, (B) Decapper to open and close 48 cryovials simultaneously (C) Tubing pump mediating the transport of the virus containing solution from the virus supply bottle to the trough on the worktable, (D) at the back: ESD-system consisting of 6 ultrasound sensors integrated in the bridge for detecting the liquid level inside the tubes and the cap status; at the front: 48-way tube rack with 48 opened cryovials.</p

Parallel performed titration assays to demonstrate the stability of the pseudovirus stock at RT and to determine the 3-fold acceptance limit between directly frozen viruses and the ones incubated at RT.

<p>Parallel performed titration assays to demonstrate the stability of the pseudovirus stock at RT and to determine the 3-fold acceptance limit between directly frozen viruses and the ones incubated at RT.</p

Precision of automatically aliquoted HIV-1 pseudovirus stocks in large-scale of low, middle and high titer viruses.

<p>Comparison of the dilution at an RLU of 150,000 to the manually aliquoted reference virus with the pre-defined 3-fold range as pass criterion.</p