Summary The lectin pathway of complement is activated upon binding of mannan‐binding lectin (MBL) or ficolins (FCNs) to their targets. Upon recognition of targets, the MBL‐and FCN‐associated serine proteases (MASPs) are activated, allowing them to generate the C3 convertase C4b2a. Recent findings indicate that the MASPs also activate components of the coagulation system. We have previously shown that MASP‐1 has thrombin‐like activity whereby it cleaves and activates fibrinogen and factor XIII. MASP‐2 has factor Xa‐like activity and activates prothrombin through cleavage to form thrombin. We now report that purified L‐FCN‐MASPs complexes, bound from serum to N‐acetylcysteine‐Sepharose, or MBL‐MASPs complexes, bound to mannan‐agarose, generate clots when incubated with calcified plasma or purified fibrinogen and factor XIII. Plasmin digestion of the clot and analysis using anti‐D‐dimer antibodies revealed that the clot was made up of fibrin and was similar to that generated by thrombin in normal human plasma. Fibrinopeptides A and B (FPA and FPB, respectively) were released after fibrinogen cleavage by L‐FCN‐MASPs complexes captured on N‐acetylcysteine‐Sepharose. Studies of inhibition of fibrinopeptide release indicated that the dominant pathway for clotting catalysed by the MASPs is via MASP‐2 and prothrombin activation, as hirudin, a thrombin inhibitor that does not inhibit MASP‐1 and MASP‐2, substantially inhibits fibrinopeptide release. In the light of their potent chemoattractant effects on neutrophil and fibroblast recruitment, the MASP‐mediated release of FPA and FPB may play a role in early immune activation. Additionally, MASP‐catalysed deposition and polymerization of fibrin on the surface of micro‐organisms may be protective by limiting the dissemination of infection.

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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...