A unique algorithm for the determination of peptide-carrier protein conjugation ratio by amino acid analysis using intrinsic internal standard.

An N-terminal peptide of the HIV-1 fusion peptide (FP) with eight amino acid residues (FP8) was conjugated to a recombinant Tetanus Toxoid Heavy Chain Fragment C (rTTHc) as a carrier protein to help boosting immunogenicity against HIV-1. In this rapid communication, a unique algorithm to determine FP-rTTHc conjugation ratio was developed based off the amino acid analysis. Five well recovered amino acids (present in both FP and rTTHc) were used to calculate the conjugation ratio, while proline (present only in rTTHc) was identified and utilized as the intrinsic internal standard for normalization. With this calculation, the assay variability was minimized (<20%), especially for conjugates with moderate to low conjugation ratios as being compared to previously reported methods. The approach offers a reliable tool to determine the efficiency of the conjugation reactions for in-process monitoring and for final conjugate product characterization.

Preclinical Development of a Fusion Peptide Conjugate as an HIV Vaccine Immunogen.

The vaccine elicitation of broadly neutralizing antibodies against HIV-1 is a long-sought goal. We previously reported the amino-terminal eight residues of the HIV-1-fusion peptide (FP8) - when conjugated to the carrier protein, keyhole limpet hemocyanin (KLH) - to be capable of inducing broadly neutralizing responses against HIV-1 in animal models. However, KLH is a multi-subunit particle derived from a natural source, and its manufacture as a clinical product remains a challenge. Here we report the preclinical development of recombinant tetanus toxoid heavy chain fragment (rTTHC) linked to FP8 (FP8-rTTHC) as a suitable FP-conjugate vaccine immunogen. We assessed 16 conjugates, made by coupling the 4 most prevalent FP8 sequences with 4 carrier proteins: the aforementioned KLH and rTTHC; the H. influenzae protein D (HiD); and the cross-reactive material from diphtheria toxin (CRM197). While each of the 16 FP8-carrier conjugates could elicit HIV-1-neutralizing responses, rTTHC conjugates induced higher FP-directed responses overall. A Sulfo-SIAB linker yielded superior results over an SM(PEG)2 linker but combinations of carriers, conjugation ratio of peptide to carrier, or choice of adjuvant (Adjuplex or Alum) did not significantly impact elicited FP-directed neutralizing responses in mice. Overall, SIAB-linked FP8-rTTHC appears to be a promising vaccine candidate for advancing to clinical assessment.