Role of lipid structure in the humoral immune response in mice to covalent lipid–peptides from the membrane proximal region of HIV-1 gp41

Abstract

The membrane proximal region (MPR) of HIV-1 gp41 is a desirable target for development of a vaccine that elicits neutralizing antibodies since the patient-derived monoclonal antibodies, 2F5 and 4E10, bind to the MPR and neutralize primary HIV isolates. The 2F5 and 4E10 antibodies crossreact with lipids and structural studies suggest that MPR immunogens may be presented in a membrane environment. We hypothesized that covalent attachment of lipid anchors would enhance the humoral immune response to MPR-derived peptides presented in liposomal bilayers. In a comparison of eight lipids conjugated to an extended 2F5 epitope peptide, a sterol, cholesterol hemisuccinate (CHEMS), was found to promote the strongest anti-peptide IgG titers (6.4 × 10 4 ) in sera of BALB/C mice. Two lipid anchors, palmitic acid and phosphatidylcholine, failed to elicit a detectable serum anti-peptide IgG response. Association with the liposomal vehicle contributed to the ability of a lipopeptide to elicit anti-peptide antibodies, but no other single factor, such as position of the lipid anchor, peptide helical content, lipopeptide partition coefficient, or presence of phosphate on the anchor clearly determined lipopeptide potency. Conjugation to CHEMS also rendered a 4E10 epitope peptide immunogenic (5.6 × 10 2 IgG titer in serum). Finally, attachment of CHEMS to a peptide spanning both the 2F5 and 4E10 epitopes elicited serum IgG antibodies that bound to each of the individual epitopes as well as to recombinant gp140. Further research into the mechanism of how structure influences the immune response to the MPR may lead to immunogens that could be useful in prime-boost regimens for focusing the immune response in an HIV vaccine.

Ablation of the Complementarity-Determining Region H3 Apex of the Anti-HIV-1 Broadly Neutralizing Antibody 2F5 Abrogates Neutralizing Capacity without Affecting Core Epitope Binding

“…Indeed, quite significant differences in the binding affinities of the native 2F5 Fab and its CDR H3 mutants are observed for the extended epitope peptide 2F5preTM, both in solution and in a liposome environment. Taken together, our results and prior reports of weak interactions between 2F5 and both of these components individually (2,18,26,28,42,56) allow us to hypothesize that the necessity of the apex of the elongated CDR H3 loop for 2F5 neutralization is caused by secondary interactions of much weaker affinity to either C-terminal MPER residues or components of a membrane bilayer or both, in the context of the energetically dominant core epitope binding. Indeed, a dual interaction of the extended CDR H3 of 2F5 with both membrane surfaces and C-terminally located gp41 MPER residues is feasible, since interfacial hydrophobicity is mainly based on aromatic and leucine residues, which can also contribute to establishing interactions among sequences embedded in a membrane milieu (40).…”

Antibody Response to Polyhistidine-Tagged Peptide and Protein Antigens Attached to Liposomes via Lipid-Linked Nitrilotriacetic Acid in Mice

“…The greater serum antibody titers induced by covalent antigen attachment could be explained by a benefit in antigen processing downstream of particulate association and internalization or by stimulation of innate pattern recognition receptors (38,46). Indeed, we have found that the structure of the lipid anchor dramatically affected the serum IgG response to liposomal N-MPR lipopeptides despite complete retention of all antigens in the liposome formulation (47).…”

“…Attachment of CHEMS to N-MPR was accomplished via amidation of a carboxylated lipid and a deprotected lysyl ε amine at the C terminus as described previously (47). Essentially, CHEMS (270 mol) was activated with 270 mol each of HBTU, HOBT, and diisopropylethylamine (DIEA) in anhydrous DMF-DCM (DCM as needed for lipid solubilization) for 30 min at room temperature, followed by addition of 67.5 mol resin and continued reaction under argon for 24 h at room temperature.…”

“…First, it oriented the peptide in a manner that mimics the native sequence, wherein the C terminus of the sequence is tethered to the membrane and the N terminus extends outward (31). Second, attachment of N-MPR peptide to liposomes via an ε-lysyl amine permitted the structure to most closely resemble that of a previously synthesized lipid-anchored control peptide found to elicit high antipeptide titers when administered in liposomal formulations to BALB/c mice (47,48).…”

Rational Design of Membrane Proximal External Region Lipopeptides Containing Chemical Modifications for HIV-1 Vaccination

“…The Szoka laboratory previously reported MPER-derived lipopeptides that are potently immunogenic when presented in lipid bilayer vesicles (50,51). In the present study, we generated a series of MPER lipopeptide immunogens bearing anionic side chain modifications that could mimic hydrophilic phospholipid head groups or inflammation-associated PTMs.…”