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An effective HIV-1 vaccine must elicit broadly neutralizing antibodies (bnAbs) against highly diverse Envelope glycoproteins (Env). Since Env with the longest hypervariable (HV) loops is more resistant to the cognate bnAbs than Env with shorter HV loops, we redesigned hypervariable loops for updated Env consensus sequences of subtypes B and C and CRF01_AE. Using modeling with AlphaFold2, we reduced the length of V1, V2, and V5 HV loops while maintaining the integrity of the Env structure and glycan shield, and modified the V4 HV loop. Spacers are designed to limit strain-specific targeting. All updated Env are infectious as pseudoviruses. Preliminary structural characterization suggests that the modified HV loops have a limited impact on Env's conformation. Binding assays show improved binding to modified subtype B and CRF01_AE Env but not to subtype C Env. Neutralization assays show increases in sensitivity to bnAbs, although not always consistently across clades. Strikingly, the HV loop modification renders the resistant CRF01_AE Env sensitive to 10-1074 despite the absence of a glycan at N332., (© 2024. The Author(s).)

Identifying whether viral features present in acute HIV-1 infection predetermine the development of neutralization breadth is critical to vaccine design. Incorporating such features in vaccine antigens could initiate cross-reactive antibody responses that could sufficiently protect vaccinees from HIV-1 infection despite the uniqueness of each founder virus. To understand the relationship between Env determinants and the development of neutralization breadth, we focused on 197 individuals enrolled in two cohorts in Thailand and East Africa (RV144 and RV217) and followed since their diagnosis in acute or early HIV-1 infection. We analyzed the distribution of variable loop lengths and glycans, as well as the predicted density of the glycan shield, and compared these envelope features to the neutralization breadth data obtained 3 years after infection ( n   = 121). Our study revealed limited evidence for glycan shield features that associate with the development of neutralization breadth. While the glycan shield tended to be denser in participants who subsequently developed breadth, no significant relationship was found between the size of glycan holes and the development of neutralization breadth. The parallel analysis of 3,000 independent Env sequences showed no evidence of directional evolution of glycan shield features since the beginning of the epidemic. Together, our results highlight that glycan shield features in acute and early HIV-1 infection may not play a role determinant enough to dictate the development of neutralization breadth and instead suggest that the glycan shield's reactive properties that are associated with immune evasion may have a greater impact. IMPORTANCE A major goal of HIV-1 vaccine research is to design vaccine candidates that elicit potent broadly neutralizing antibodies (bNAbs). Different viral features have been associated with the development of bNAbs, including the glycan shield on the surface of the HIV-1 Envelope (Env). Here, we analyzed data from two cohorts of individuals who were followed from early infection to several years after infection spanning multiple HIV-1 subtypes. We compared Env glycan features in HIV-1 sequences obtained in early infection to the potency and breadth of neutralizing antibodies measured 1 to 3 years after infection. We found limited evidence of glycan shield properties that associate with the development of neutralization breadth in these cohorts. These results may have important implications for antigen design in future vaccine strategies and emphasize that HIV-1 vaccines will need to rely on a complex set of properties to elicit neutralization breadth.

The envelope glycoprotein (Env) of the human immunodeficiency virus (HIV), has been the primary target for the development of a protective vaccine against infection. The extensive N-linked glycosylation on Env is an important consideration as it may affect efficacy, stability, and expression yields. The expression host has been shown to influence the extent and type of glycosylation that decorates the protein target. Here, we report the glycosylation profile of the candidate subtype C immunogen CO6980v0c22 gp145, which is currently in Phase I clinical trials, produced in two different host cells: CHO-K1 and Expi293F. The amino acid sequence for both glycoproteins was confirmed to be identical by peptide mass fingerprinting. However, the isoelectric point of the proteins differed; 4.5-5.5 and 6.0-7.0 for gp145 produced in CHO-K1 and Expi293F, respectively. These differences in pI were eliminated by enzymatic treatment with sialidase, indicating a large difference in the incorporation of sialic acid between hosts. This dramatic difference in the number of sialylated glycans between hosts was confirmed by analysis of PNGase F-released glycans using MALDI-ToF MS. These differences in glycosylation, however, did not greatly translate into differences in antibody recognition. Biosensor assays showed that gp145 produced in CHO-K1 had similar affinity toward the broadly neutralizing antibodies, 2G12 and PG16, as the gp145 produced in Expi293F. Additionally, both immunogens showed the same reactivity against plasma of HIV-infected patients. Taken together, these results support the notion that there are sizeable differences in the glycosylation of Env depending on the expression host. How these differences translate to vaccine efficacy remains unknown., Competing Interests: The authors have declared that no competing interests exist.

The V1V2 region of the HIV-1 envelope is the target of several broadly neutralizing antibodies (bNAbs). Antibodies to V1V2 elicited in the RV144 clinical trial correlated with a reduced risk of HIV infection, but these antibodies were without broad neutralizing activity. Antibodies targeting V1V2 also correlated with a reduced viral load in immunized macaques challenged with simian immunodeficiency virus (SIV) or simian/human immunodeficiency virus (SHIV). To focus immune responses on V1V2, we engrafted the native, glycosylated V1V2 domain onto five different multimeric scaffold proteins and conducted comparative immunogenicity studies in macaques. Vaccinated macaques developed high titers of plasma and mucosal antibodies that targeted structurally distinct V1V2 epitopes. Plasma antibodies displayed limited neutralizing activity but were functionally active for ADCC and phagocytosis, which was detectable 1-2 years after immunizations ended. This study demonstrates that multivalent, glycosylated V1V2-scaffold protein immunogens focus the antibody response on V1V2 and are differentially effective at inducing polyfunctional antibodies with characteristics associated with protection., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Eliciting broadly reactive functional antibodies remains a challenge in human immunodeficiency virus type 1 (HIV-1) vaccine development that is complicated by variations in envelope (Env) subtype and structure. The majority of new global HIV-1 infections are subtype C, and novel antigenic properties have been described for subtype C Env proteins. Thus, an HIV-1 subtype C Env protein (CO6980v0c22) from an infected person in the acute phase (Fiebig stage I/II) was developed as a research reagent and candidate immunogen. The gp145 envelope is a novel immunogen with a fully intact membrane-proximal external region (MPER), extended by a polylysine tail. Soluble gp145 was enriched for trimers that yielded the expected "fan blade" motifs when visualized by cryoelectron microscopy. CO6980v0c22 gp145 reacts with the 4E10, PG9, PG16, and VRC01 HIV-1 neutralizing monoclonal antibodies (MAbs), as well as the V1/V2-specific PGT121, 697, 2158, and 2297 MAbs. Different gp145 oligomers were tested for immunogenicity in rabbits, and purified dimers, trimers, and larger multimers elicited similar levels of cross-subtype binding and neutralizing antibodies to tier 1 and some tier 2 viruses. Immunized rabbit sera did not neutralize the highly resistant CO6980v0c22 pseudovirus but did inhibit the homologous infectious molecular clone in a peripheral blood mononuclear cell (PBMC) assay. This Env is currently in good manufacturing practice (GMP) production to be made available for use as a clinical research tool and further evaluation as a candidate vaccine., Importance: At present, the product pipeline for HIV vaccines is insufficient and is limited by inadequate capacity to produce large quantities of vaccine to standards required for human clinical trials. Such products are required to evaluate critical questions of vaccine formulation, route, dosing, and schedule, as well as to establish vaccine efficacy. The gp145 Env protein presented in this study forms physical trimers, binds to many of the well-characterized broad neutralizing MAbs that target conserved Env epitopes, and induce cross-subtype neutralizing antibodies as measured in both cell line and primary cell assays. This subtype C Env gp145 protein is currently undergoing good manufacturing practice production for use as a reagent for preclinical studies and for human clinical research. This product will serve as a reagent for comparative studies and may represent a next-generation candidate HIV immunogen., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)