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.The fusion peptide (FP) site of vulnerability on the HIV-1 envelope (Env) glycoprotein has recently been shown to be a promising vaccine target 1-3 . FP, a hydrophobic region of ~15 residues at the N terminus of the gp41 transmembrane glycoprotein, is an essential component of the HIV entry machinery 4 . FP embeds in the target cell membrane during the pre-hairpin intermediate stage of entry, where it serves to anchor the rearranging viral spike and to facilitate the merging of viral and cell membranes. The N-terminal portion of FP is solvent accessible and recognized by broadly neutralizing antibodies PGT151 5,6 , N123-VRC34.01 3 , and ACS202 7 . Because FP is a short linear peptide, it has low inherent immunogenicity due to its lack of helper T cell epitopes. Coupling peptides to highly immunogenic carrier proteins is a well-established approach for providing T cell help to peptide immunogens [8][9][10][11] . When the N-terminal 6-10 residues of FP are coupled to keyhole limpet hemocyanin (KLH), a standard protein carrier widely used in biotechnology, the resultant FP-KLH conjugate immunogens are able to induce broadly neutralizing FP-directed immune responses in mice, guinea pigs, and rhesus macaques 1,2,12 . Vaccine-induced FP-directed antibodies from mice or NHP neutralize up to 31% or 59%, respectively, of a cross-clade panel of 208 HIV-1 strains 2 .These results (illustrated in Fig. 1a) indicate FP coupled to a carrier protein to be a promising candidate immunogen. However, KLH is a multi-subunit metalloprotein derived from natural sources 13-15 with both sequence and glycan heterogeneity, which pose manufacturin...

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High throughput process development (HTPD) using liquid handling robotics and RoboColumns is an established methodology in downstream process development to screen chromatography resins and optimize process designs to meet target product profiles. However, HTPD is not yet widely available for use in viral clearance capability of the resin due to a variety of constraints. In the present study, a BSL‐1‐compatible, non‐infectious MVM model, MVM‐VLP, was tested for viral clearance assessment with various resin and membrane chromatography operations in a HTPD mode. To detect the MVM‐VLP in the high throughput experiments, an electrochemiluminescence immunoassay (ECLIA) assay was developed with up to 5 logs of dynamic range. Storage time suitability of MVM‐VLP solutions in various buffer matrices, in the presence or absence of a glycoprotein vaccine candidate, were assessed. Then, MVM‐VLP and a test article monoclonal antibody (mAb) were used in a HTPD design that included commercially available ion exchange media chemistries, elucidating a wide variety of viral clearance ability at different operating conditions. The methodologies described herein have the potential to be a part of the process design stage in biologics manufacturing process development, which in turn can reduce risk associated with viral clearance validation studies.