GoKawiilAnimals
The animal work was approved by the Emory University Institutional Animal Care and Use Committee (IACUC) under protocol 202100136.
Twelve adult Indian-origin rhesus macaques (Macaca mulatta) (RM) were housed at the Emory National Primate Research Center (ENPRC) and maintained in accordance with NIH guidelines. Animal care facilities are accredited by the US Department of Agriculture (USDA) and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. Animals were treated with anaesthesia (ketamine, 5–10 mg per kg; or telazol, 3–6 mg per kg) and analgesics for procedures including intramuscular (i.m.) and subcutaneous (s.c.) immunization, and blood draws as per veterinarian recommendations and IACUC approved protocols. Rhesus macaques were male and female, aged 3–4 years at the start of the study, with an average weight of 4.8 kg. Animals were grouped to divide age and weight as evenly as possible between the groups receiving either soluble or liposome-conjugated trimers. Animals were housed in pairs for the duration of the study. For the immunization group size, we established the sample size primarily by extensive historical experience in immunization studies, which indicates that the assignment of six animals per immunization group will result in the detection of significant differences in antibody production among different experimental groups. Moreover, we have taken into consideration the three Rs (replacement, reduction and refinement) principle that guides the humane use of animals in scientific research; in particular, we considered reduction, which recommends using the fewest animals necessary to achieve valid experimental results.
Cell lines
HEK293F cells were cultured in FreeStyle 293 Expression Medium (Thermo Fisher Scientific) supplemented with 1× antibiotic–antimycotic solution (Sigma-Aldrich) in a humidified incubator (125 rpm, 8% CO 2 , 37 °C). TZM-bl and HEK293T cells were cultured in Dulbecco’s modified Eagle medium (cDMEM) supplemented with 10% fetal BSA and 1× penicillin–streptomycin–glutamine (Gibco) at 5% CO 2 and 37 °C. Expi293F cells were cultured in medium from 293F Expression System Kit in a humidified incubator (125 rpm, 8% CO 2 , 37 °C).
Design of NFL trimer immunogens and probes
The Env sequences used for the generation of the NFL trimeric proteins were derived from the following HIV-1 viral sequences obtained from the Los Alamos National Laboratory HIV sequence database: Q23.17 (AF0048885.1), ZM233M.6 (DQ388517), WITO4160.33 (AY835451), 001428_2 (EF117266), 92RW020 (AY669706), BG505.W6M (DQ208456) and 16055_2 (EF117268). These DNA sequences were codon optimized to enhance protein expression and modified as follows to make stabilized NFL trimeric Env proteins: the natural HIV-1 leader sequence was replaced by a CD5 leader sequence. The four-residue furin cleavage site 508-REKR-511 was substituted with a ten-amino-acid flexible linker comprising the sequence G 4 SG 4 S28. The C terminus of the Env sequence was truncated at residue 664, resulting in the elimination of the membrane proximal external proximal region, transmembrane domain and cytoplasmic tail41. An additional linker comprising the residues GGGGSHHHHHHHHGSGC was added to the C terminus to facilitate coupling to liposomes through the terminal cysteine residue. Finally, a series of stabilizing mutations was introduced to create highly stable and homogeneous trimeric proteins with near-native conformation. These stabilizing mutations consist of the TD (BG505-trimer-derived residues), helix-breaking glycine and proline substitutions, unnatural disulfides and V3-loop and fusion-peptide-stabilizing mutations29,30,32.
Trimer expression, purification, characterization and trimer-liposome preparation
The expression and purification protocols for HIV-1 Env NFL trimers were described in detail previously28,30,42,43. NFL trimers used as immunogens were expressed transiently in 293F cells and purified over a lectin column followed by negative selection using the non-neutralizing mAb F105 followed by size-exclusion chromatography (SEC). The trimers were characterized for their structural conformation and antigenicity by SEC, BLI and DSC. In brief, the BLI analysis was carried out on an Octet Red instrument (Sartorius) with IgGs immobilized on anti-human IgG Fc capture sensors (Sartorius). The Env trimers were assessed as free analytes in solution (PBS pH 7.4) at a final concentration of 250 nM. Association and dissociation were measured for 60 s respectively. The data were analysed using ForteBio software v.11.1 and kinetic parameters were obtained using a global 1:1 fit model. The thermal transition temperature (T m ) of the NFL trimers was determined by DSC using a MicroCal VP-Capillary DSC instrument (Malvern Panalytics). The trimer samples were dialysed in PBS, pH 7.4, and 400 μl of the trimer sample at concentration of 0.25 mg ml−1 was loaded into the instrument. The dialysis buffer was used as the reference solution. The DSC experiments were performed at a scanning rate of 1 K min−1 under 3.0 atmospheres of pressure. The data were analysed after buffer correction, normalization and baseline subtraction using MicroCal VP-Capillary DSC analysis software provided by the manufacturer (Origin 7 SR4 v.7.0522). Liposome-conjugated trimers were used as immunogens in NHPs Q7–12. The conjugation protocol for generating trimer-liposomes was described in detail previously31. Liposomes comprised 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl) cyclohexane-carboxamide]31. After chemical conjugation with the NFL trimers, the trimer-liposomes were further purified over the Superdex 200 Increase 10/300 GL column in PBS pH 7.4 buffer to remove unconjugated trimers from the trimer-liposomes. The conjugation of trimer to liposomes was confirmed by imaging the trimer-liposomes using negative-stain electron microscopy (Scripps Research, EM core).
Calcium Flux
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