Production of a Short Recombinant C4V3 HIV-1 Immunogen That Induces Strong Anti-HIV Responses by Systemic and Mucosal Routes Without the Need of Adjuvants

Abstract

Synthetic peptides have been shown to evoke neutralizing and cytotoxic protective anti-HIV responses in mice and other animal models. Recent data support that C4V3 peptides can induce anti- V3 antibodies that neutralize primary isolates. Critical to the success of peptide-based vaccines is the development of strategies to augment their immunogenicity while reducing their large-scale production costs. Therefore, finding efficient and economical alternatives for the production of epitopic vaccines could have an impact on researches using such immunogens. Herein, we report the recombinant production and immunological characterization of a short polypeptide which carries the three relevant epitopes contained in a C4V3 peptide. This polypeptide, named rC4V3, was efficiently produced in E. coli, yielding more than 75 mg per culture liter. No major difficulties were found in the recovery, refolding and purification of this peptide; the latter facilitated by C-terminal inclusion of a histidine tag. The immunogenicity of this protein was studied by administering it intramuscularly or intranasally to mice and it demonstrated to be a strong elicitor of anti-HIV antibodies at systemic and mucosal compartments. Remarkably, such responses were attained with rC4V3 even without the need of adjuvants. We can conclude that this protein might be a promising tool for studies using epitope-based vaccine designs.

A chloroplast-derived C4V3 polypeptide from the human immunodeficiency virus (HIV) is orally immunogenic in mice

“…5a-c). However, the molecular weight of the immunoreactive protein was higher than expected, as previously noted by Varona-Santos et al (2006). These results suggested that tobacco chloroplasts were capable of expressing the C4V3 and retaining its antigenic determinants.…”

“…Previously, we have reported on the production of the C4V3 protein at high levels in recombinant E. coli, demonstrating that despite its low molecular weight, the recombinant C4V3 could induce strong humoral immune responses against HIV-1 sequences when delivered by systemic and mucosal routes, even without the aid of adjuvants (Varona-Santos et al 2006). In this study, we have engineered tobacco plants to produce a plastidexpressed C4V3 protein.…”

“…The C4V3 encoding gene has been previously described by Varona-Santos et al (2006). This gene comprises a V3 loop from the MN isolate as well as a C4 peptide fused to the N terminus of V3 along with a His tag at the carboxi terminus.…”

“…For immunization, a pure antigen (400 lg) was produced as described by Varona-Santos et al (2006), and administered by the subcutaneous (sc) route in six doses. The rabbit was primed with a dose of 400 lg of rC4V3 in 200 ll of PBS plus 200 ll of complete Freund's adjuvant, and then subjected to 5 weekly doses consisting of 400 lg of rC4V3 plus 200 ll of incomplete Freund's adjuvant.…”

“…Blood samples were incubated at 25°C for 1 h to facilitate clotting, and then centrifuged at 5,000 rpm for 5 min at 4°C. The anti-serum was collected, and immunoreactivity was assessed by both Western blot and ELISA using plates coated with pure rC4V3 (Varona-Santos et al 2006). …”