HIV-1 Neutralization Profile and Plant-Based Recombinant Expression of Actinohivin, an Env Glycan-Specific Lectin Devoid of T-Cell Mitogenic Activity

The development of a topical microbicide blocking the sexual transmission of HIV-1 is urgently needed to control the global HIV/AIDS pandemic. The actinomycete-derived lectin actinohivin (AH) is highly specific to a cluster of high-mannose-type glycans uniquely found on the viral envelope (Env). Here, we evaluated AH's candidacy toward a microbicide in terms of in vitro anti-HIV-1 activity, potential side effects, and recombinant producibility. Two validated assay systems based on human peripheral blood mononuclear cell (hPBMC) infection with primary isolates and TZM-bl cell infection with Env-pseudotyped viruses were employed to characterize AH's anti-HIV-1 activity. In hPMBCs, AH exhibited nanomolar neutralizing activity against primary viruses with diverse cellular tropisms, but did not cause mitogenicity or cytotoxicity that are often associated with other anti-HIV lectins. In the TZM-bl-based assay, AH showed broad anti-HIV-1 activity against clinically-relevant, mucosally transmitting strains of clades B and C. By contrast, clade A viruses showed strong resistance to AH. Correlation analysis suggested that HIV-1′s AH susceptibility is significantly linked to the N-glycans at the Env C2 and V4 regions. For recombinant (r)AH expression, we evaluated a tobacco mosaic virus-based system in Nicotiana benthamiana plants as a means to facilitate molecular engineering and cost-effective mass production. Biochemical analysis and an Env-mediated syncytium formation assay demonstrated high-level expression of functional rAH within six days. Taken together, our study revealed AH's cross-clade anti-HIV-1 activity, apparent lack of side effects common to lectins, and robust producibility using plant biotechnology. These findings justify further efforts to develop rAH toward a candidate HIV-1 microbicide

Anti-HIV-1 activity of AH against Env-pseudotyped viruses in TZM-bl cells.

<p>*IC₅₀s exceeding the highest concentrations of samples tested in the assay are expressed as >25 or >50 µg/ml. ND: not determined.</p

Activity analysis of <i>N. benthamiana</i>-expressed rAH.

<p>(A) The gp120-binding capacity of rAH expressed in <i>N. benthamiana</i> was evaluated by gp120-ELISA using AH-expressing and control (uninfiltrated) leaf extracts prepared as described in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011143#s2" target="_blank">Methods</a>. Results of a representative sample obtained from a ∼2 g batch of leaf materials are shown. Data points are the means ± SD of triplicate analysis. This analysis was also used to determine the quantity of rAH in the extract based on a standard curve created by actinomycete-derived purified AH. (B) X-gal staining of syncytia formation by HeLa/Env/tat and HeLa/CD4/LacZ. The two cell lines were incubated for 18 h at 37°C with: <b>a</b>, media alone; <b>b</b>, 1/20-diluted non-infiltrated control leaf sample; <b>c</b>, 1 µM actinomycete-derived AH; and <b>d</b>, 1/20-diluted rAH-expressing leaf sample (containing approximately 0.32 µM, or 4.0 µg/ml, of rAH, as determined by gp120-ELISA). (C) Quantitative analysis of syncytia formation by HeLa/Env/tat and HeLa/CD4/LacZ. The two cell lines were incubated for 18 h at 37°C with serially diluted control or rAH-expressing leaf samples. See Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011143#s2" target="_blank">Methods</a> for the assay detail. Differences between the samples at each dilution point were evaluated by two-way analysis of variance followed by Bonferroni posttests (*** <i>p</i><0.001).</p

Neutralization of primary HIV-1 isolates in hPBMCs.

<p>(A) The hPBMC-based neutralization assay. The reduction of p24 gag protein production in hPBMCs was measured as an endpoint. hPBMCs (pooled from four different donors) were infected with R5 B clade (SF162), R5 C clade (ZA/97/009), X4 B clade (HT/92/599), or R5X4 B clade (BZ167) in the presence of various concentrations of AH. Samples were analyzed in quadruplicate.Results are expressed as mean ± SEM. See Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011143#s2" target="_blank">Methods</a> for assay details. Dose-dependent neutralization was observed for all viruses. (B) Cytotoxicity analysis. Potential toxicity of AH in hPBMCs was analyzed based on the LDH activity in the culture medium after incubation of hPBMCs with various concentrations of AH. Analysis was performed in quadruplicate in two different pools of hPBMCs. Results are expressed as mean ± SEM. The lack of hPMBC cytotoxicity was demonstrated for up to 50 µg/ml of AH.</p

Schematic representation of Env and potential NLG positions in selected AH-susceptible and -resistant viruses.

<p>Constant regions (C1–C5; shown in white) and variable regions (V1–V5; shown in grey) are shown for the two most susceptible viruses (SS1196.1 and TV1.21) and two highly resistant viruses (ZM109F.PB4 and Q259.d2.17) to AH, according to the results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011143#pone-0011143-t001" target="_blank">Table 1</a>. Black boxes indicate deletions in the corresponding regions. Asterisks represent the position of sequons. Potential targets of AH within the C2 and V4 regions (those present in both the susceptible viruses but absent in either of the resistant strains) are indicated by arrows.</p

Expression of rAH in <i>N. benthamiana</i>.

<p>(A) Representative TMV vector-infiltrated <i>N. benthamiana</i> leaves at 6 dpi. The leaves show only a minor level of tissue necrosis. (B) Electrophoretic analysis of rAH expression in leaf extract. Coomassie-stained SDS-PAGE gel (Lanes 1–3) and western blot analysis (Lanes 4–7) are shown. Total leaf proteins were extracted in a buffer containing 2% SDS and separated under reducing conditions. Lanes 1 and 4: non-infiltrated control leaf extract; lanes 2, 3, 5, and 6: vector-infiltrated leaf extracts made from two independent infiltration events; and lane 7: the dialyzed aqueous fraction of infiltrated leaf proteins prepared by 6 M guanidine buffer extraction. Asterisks indicate the bands corresponding to rAH (Mw: 12.5 kDa). Efficient extraction of gp120-binding rAH required a buffer containing a chaotropic agent such as SDS or guanidine HCl. The fraction contains apparent multimers of rAH (Lanes 5 and 6). These aggregates are largely absent after dialysis, while the monomer protein stays soluble (Lane 7). See text for detail.</p

Correlation between the number of NLG sequons and AH susceptibility in Env-pseudotyped viruses.

<p>For each virus tested in the TZM-bl-based assay, the number of NLG sites (i.e., sequons) in the indicated Env region was plotted against the IC₅₀. The IC₅₀ values of >25 µg/ml were approximated to 50 µg/ml. Correlation was analyzed by the non-parametric Spearman's correlation coefficient. The correlation coefficient (r) and <i>p</i> values are shown in the box in each graph. A <i>p</i> value of less than 0.05 is regarded as statistically significant. Linear regression analysis was used to display a best fit line to the data.</p