sCD4-17b bifunctional protein: Extremely broad and potent neutralization of HIV-1 Env pseudotyped viruses from genetically diverse primary isolates

<p>Abstract</p> <p>Background</p> <p>We previously described a potent recombinant HIV-1 neutralizing protein, sCD4-17b, composed of soluble CD4 attached via a flexible polypeptide linker to an SCFv of the 17b human monoclonal antibody directed against the highly conserved CD4-induced bridging sheet of gp120 involved in coreceptor binding. The sCD4 moiety of the bifunctional protein binds to gp120 on free virions, thereby enabling the 17b SCFv moiety to bind and block the gp120/coreceptor interaction required for entry. The previous studies using the MAGI-CCR5 assay system indicated that sCD4-17b (in concentrated cell culture medium, or partially purified) potently neutralized several genetically diverse HIIV-1 primary isolates; however, at the concentrations tested it was ineffective against several other strains despite the conservation of binding sites for both CD4 and 17b. To address this puzzle, we designed variants of sCD4-17b with different linker lengths, and tested the neutralizing activities of the immunoaffinity purified proteins over a broader concentration range against a large number of genetically diverse HIV-1 primary isolates, using the TZM-bl Env pseudotype assay system. We also examined the sCD4-17b sensitivities of isogenic viruses generated from different producer cell types.</p> <p>Results</p> <p>We observed that immunoaffinity purified sCD4-17b effectively neutralized HIV-1 pseudotypes, including those from HIV-1 isolates previously found to be relatively insensitive in the MAGI-CCR5 assay. The potencies were equivalent for the original construct and a variant with a longer linker, as observed with both pseudotype particles and infectious virions; by contrast, a construct with a linker too short to enable simultaneous binding of the sCD4 and 17b SCFv moieties was much less effective. sCD4-17b displayed potent neutralizing activity against 100% of nearly 4 dozen HIV-1 primary isolates from diverse genetic subtypes (clades A, B, C, D, F, and circulating recombinant forms AE and AG). The neutralization breadth and potency were superior to what have been reported for the broadly neutralizing monoclonal antibodies IgG b12, 2G12, 2F5, and 4E10. The activity of sCD4-17b was found to be similar against isogenic virus particles from infectious molecular clones derived either directly from the transfected producer cell line or after a single passage through PBMCs; this contrasted with the monoclonal antibodies, which were less potent against the PMBC-passaged viruses.</p> <p>Conclusions</p> <p>The results highlight the extremely potent and broad neutralizing activity of sCD4-17b against genetically diverse HIV-1 primary isolates. The bifunctional protein has potential applications for antiviral approaches to combat HIV infection.</p

Robust vaginal colonization of macaques with a novel vaginally disintegrating tablet containing a live biotherapeutic product to prevent HIV infection in women.

MucoCept is a biotherapeutic for prevention of HIV-1 infection in women and contains a human, vaginal Lactobacillus jensenii that has been genetically enhanced to express the HIV-1 entry inhibitor, modified cyanovirin-N (mCV-N). The objective of this study was to develop a solid vaginal dosage form that supports sustained vaginal colonization of the MucoCept Lactobacillus at levels previously shown, with freshly prepared cultures, to protect macaques from SHIV infection and to test this formulation in a macaque vaginal colonization model. Vaginally disintegrating tablets were prepared by lyophilizing the formulated bacteria in tablet-shaped molds, then packaging in foil pouches with desiccant. Disintegration time, potency and stability of the tablets were assessed. For colonization, non-synchronized macaques were dosed vaginally with either one tablet or five tablets delivered over five days. Vaginal samples were obtained at three, 14, and 21 days post-dosing and cultured to determine Lactobacillus colonization levels. To confirm identity of the MucoCept Lactobacillus strain, genomic DNA was extracted from samples on days 14 and 21 and a strain-specific PCR was performed. Supernatants from bacteria were tested for the presence of the mCV-N protein by Western blot. The tablets were easy to handle, disintegrated within two minutes, potent (5.7x1011 CFU/g), and stable at 4°C and 25°C. Vaginal administration of the tablets to macaques resulted in colonization of the MucoCept Lactobacillus in 66% of macaques at 14 days post-dosing and 83% after 21 days. There was no significant difference in colonization levels for the one or five tablet dosing regimens (p=0.88 Day 14, p=0.99 Day 21). Strain-specific PCR confirmed the presence of the bacteria even in culture-negative macaques. Finally, the presence of mCV-N protein was confirmed by Western blot analysis using a specific anti-mCV-N antibody

Engineering of a Human Vaginal Lactobacillus Strain for Surface Expression of Two-Domain CD4 Molecules▿

Women are at significant risk of heterosexually transmitted human immunodeficiency virus (HIV) infection, with the mucosal epithelium of the cervix and vagina serving as a major portal of entry. The cervicovaginal mucosa naturally harbors dynamic microflora composed predominantly of lactobacilli, which may be genetically modified to serve as a more efficient protective barrier against the heterosexual transmission of HIV. We selected a vaginal strain of Lactobacillus, L. jensenii 1153, for genetic modification to display surface-anchored anti-HIV proteins. Genomic sequencing analyses revealed that L. jensenii 1153 encodes several unique high-molecular-weight cell wall-anchored proteins with a C-terminal cell wall sorting LPQTG motif. In this report, we employed these proteins to express a surface-anchored two-domain CD4 (2D CD4) molecule in L. jensenii 1153. Our studies indicated that the C-terminal cell wall sorting signal LPQTG motif alone is insufficient to drive the surface expression of heterologous proteins, and the display of surface-anchored 2D CD4 molecules required native sequences of a defined length upstream of the unique C-terminal LPQTG cell wall sorting signal and the positively charged C terminus in a Lactobacillus-based expression system. The modified L. jensenii strain displayed 2D CD4 molecules that were uniformly distributed on bacterial surfaces. The surface-anchored 2D CD4 molecule was recognized by a conformation-dependent anti-CD4 antibody, suggesting that the expressed proteins adopted a native conformation. The establishment of this Lactobacillus-based surface expression system, with potential broad applicability, represents a major step toward developing an inexpensive yet durable approach to topical microbicides for the mitigation of heterosexual transmission of HIV and other mucosally transmitted viral pathogens

Stability of MucoCept VDTs.

<p>Two batches of tablets were prepared and placed in storage at 4°C, 25°C, or 37°C. Potency was measured as CFU/g at time zero, one, two, three, six, and 12 months to assess stability. The mean initial potency of the tablets was 5.7x10¹¹ CFU/g. Error bars represent the standard error of the mean (SEM). The potency of tablets stored at 4°C and 25°C for one year was 4.9x10¹¹ CFU/g and 1.2x10¹¹ CFU/g, respectively. Tablets stored at 37°C lost 1.8 log₁₀ CFU/g of their initial potency after two months (to 9.4x10⁹ CFU/g). Stability at 37°C was discontinued after two months.</p

Vaginal colonization of macaques following administration of MucoCept VDTs.

<p>Macaques were vaginally administered one (closed symbols) or five tablets (open symbols) and colonization of <i>L</i>. <i>jensenii</i> 1153–1666 determined at days 14 and 21 post-dosing. In addition, macaques dosed with one tablet were swabbed after three days to determine the initial delivery of viable bacteria to the vagina. Colonization was expressed as CFU/swab following growth of vaginal bacterial dilutions on Rogosa agar and colony counting. Menses (M) is noted in three macaques on the day of sampling. Animals in follicular phase or luteal phase at the time of tablet administration are noted when accurate determination was possible. No significant difference was found between one tablet and five tablet administration at day 14 p = 0.88 or day 21 p = 0.99 based on a one sided Wilcoxon Rank Sum test.</p

Western blot to confirm mCV-N protein expression.

<p>Proteins in stationary phase conditioned media from <i>L</i>. <i>jensenii</i> 1153–1666 isolated from macaques that received one-tablet or five-tablets were separated on a 12% Bis-Tris gel. A representative gel is shown from the day 14-post dosing time point. The parental strain, <i>L</i>. <i>jensenii</i> 1153, is shown as the negative control, and <i>L</i>. <i>jensenii</i> 1153–1666 as the positive control. Three macaques from the five-tablet group and two macaques from the one-tablet group are shown, confirming that colonies of <i>L</i>. <i>jensenii</i> 1153–1666 recovered from the macaques continued to produce mCV-N protein.</p