Explore the vast range of titles available.

Over the last decade, multiple broadly neutralizing monoclonal antibodies (bN-mAbs) to the HIV-1 envelope protein (Env) gp120 have been described. Many of these recognize epitopes consisting of both amino acid and glycan residues. Moreover, the glycans required for binding of these bN-mAbs are early intermediates in the N-linked glycosylation pathway. This type of glycosylation substantially alters the mass and net charge of Envs compared to molecules with the same amino acid sequence but possessing mature, complex (sialic acid-containing) carbohydrates. Since cell lines suitable for biopharmaceutical production that limit N-linked glycosylation to mannose-5 (Man5) or earlier intermediates are not readily available, the production of vaccine immunogens displaying these glycan-dependent epitopes has been challenging. Here, we report the development of a stable suspension-adapted Chinese hamster ovary (CHO) cell line that limits glycosylation to Man5 and earlier intermediates. This cell line was created using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing system and contains a mutation that inactivates the gene encoding Mannosyl (Alpha-1,3-)-Glycoprotein Beta-1,2-N-Acetylglucosaminyltransferase (MGAT1). Monomeric gp120s produced in the MGAT1- CHO cell line exhibit improved binding to prototypic glycan-dependent bN-mAbs directed to the V1/V2 domain (e.g., PG9) and the V3 stem (e.g., PGT128 and 10-1074) while preserving the structure of the important glycan-independent epitopes (e.g., VRC01). The ability of the MGAT1- CHO cell line to limit glycosylation to early intermediates in the N-linked glycosylation pathway without impairing the doubling time or ability to grow at high cell densities suggests that it will be a useful substrate for the biopharmaceutical production of HIV-1 vaccine immunogens.

Several candidate HIV subunit vaccines based on recombinant envelope (Env) glycoproteins have been advanced into human clinical trials. To facilitate biopharmaceutical production, it is necessary to produce these in CHO (Chinese Hamster Ovary) cells, the cellular substrate used for the manufacturing of most recombinant protein therapeutics. However, previous studies have shown that when recombinant Env proteins from clade B viruses, the major subtype represented in North America, Europe, and other parts of the world, are expressed in CHO cells, they are proteolyzed and lack important glycan-dependent epitopes present on virions. Previously, we identified C1s, a serine protease in the complement pathway, as the endogenous CHO protease responsible for the cleavage of clade B laboratory isolates of -recombinant gp120s (rgp120s) expressed in stable CHO-S cell lines. In this paper, we describe the development of two novel CHOK1 cell lines with the C1s gene inactivated by gene editing, that are suitable for the production of any protein susceptible to C1s proteolysis. One cell line, C1s-/- CHOK1 2.E7, contains a deletion in the C1s gene. The other cell line, C1s-/- MGAT1- CHOK1 1.A1, contains a deletion in both the C1s gene and the MGAT1 gene, which limits glycosylation to mannose-5 or earlier intermediates in the N-linked glycosylation pathway. In addition, we compare the substrate specificity of C1s with thrombin on the cleavage of both rgp120 and human Factor VIII, two recombinant proteins known to undergo unintended proteolysis (clipping) when expressed in CHO cells. Finally, we demonstrate the utility and practicality of the C1s-/- MGAT1- CHOK1 1.A1 cell line for the expression of clinical isolates of clade B Envs from rare individuals that possess broadly neutralizing antibodies and are able to control virus replication without anti-retroviral drugs (elite neutralizer/controller phenotypes). The Envs represent unique HIV vaccine immunogens suitable for further immunogenicity and efficacy studies.

Non-neutralizing IgG to the V1V2 loop of HIV-1 gp120 correlates with a decreased risk of HIV-1 infection but the mechanism of protection remains unknown. This V1V2 IgG correlate was identified in RV144 Thai trial vaccine recipients, who were primed with a canarypox vector expressing membrane-bound gp120 (vCP1521) and boosted with vCP1521 plus a mixture gp120 proteins from clade B and clade CRF01_AE (B/E gp120). We sought to determine whether the mechanism of vaccine protection might involve antibody-dependent complement activation. Complement activation was measured as a function of complement component C3d deposition on V1V2-coated beads in the presence of RV144 sera. Variable levels of complement activation were detected two weeks post final boosting in RV144, which is when the V1V2 IgG correlate was identified. The magnitude of complement activation correlated with V1V2-specific serum IgG and was stronger and more common in RV144 than in HIV-1 infected individuals and two related HIV-1 vaccine trials, VAX003 and VAX004, where no protection was seen. After adjusting for gp120 IgA, V1V2 IgG, gender, and risk score, complement activation by case-control plasmas from RV144 correlated inversely with a reduced risk of HIV-1 infection, with odds ratio for positive versus negative response to TH023-V1V2 0.42 (95% CI 0.18 to 0.99, p = 0.048) and to A244-V1V2 0.49 (95% CI 0.21 to 1.10, p = 0.085). These results suggest that complement activity may have contributed in part to modest protection against the acquisition of HIV-1 infection seen in the RV144 trial.

The production of envelope glycoproteins (Envs) for use as HIV vaccines is challenging. The yield of Envs expressed in stable Chinese Hamster Ovary (CHO) cell lines is typically 10-100 fold lower than other glycoproteins of pharmaceutical interest. Moreover, Envs produced in CHO cells are typically enriched for sialic acid containing glycans compared to virus associated Envs that possess mainly high-mannose carbohydrates. This difference alters the net charge and biophysical properties of Envs and impacts their antigenic structure. Here we employ a novel robotic cell line selection strategy to address the problems of low expression. Additionally, we employed a novel gene-edited CHO cell line (MGAT1- CHO) to address the problems of high sialic acid content, and poor antigenic structure. We demonstrate that stable cell lines expressing high levels of gp120, potentially suitable for biopharmaceutical production can be created using the MGAT1- CHO cell line. Finally, we describe a MGAT1- CHO cell line expressing A244-rgp120 that exhibits improved binding of three major families of bN-mAbs compared to Envs produced in normal CHO cells. The new strategy described has the potential to eliminate the bottleneck in HIV vaccine development that has limited the field for more than 25 years.

To date, the RV144 HIV vaccine trial has been the only study to show that immunization can confer protection from HIV infection. While encouraging, the modest 31.2% (P = 0.04) efficacy achieved in this study left significant room for improvement, and created an incentive to optimize the AIDSVAX B/E vaccine immunogens to increase the level of vaccine efficacy. Since the completion of the RV144 trial, our understanding of the antigenic structure of the HIV envelope protein, gp120, and of the specificity of broadly neutralizing monoclonal antibodies (bN-mAbs) that bind to it, has significantly improved. In particular, we have learned that multiple families of bN-mAbs require specific oligomannose glycans for binding. Both of the monomeric gp120 immunogens (MN- and A244-rgp120) in the AIDSVAX B/E vaccine used in the RV144 trial were enriched for glycans containing high levels of sialic acid, and lacked critical N-linked glycosylation sites required for binding by several families of bN-mAbs. The absence of these epitopes may have contributed to the low level of efficacy achieved in this study. In this report, we describe our efforts to improve the antigenic structure of the rgp120 immunogens used in the vaccine by optimizing glycan-dependent epitopes recognized by multiple bN-mAbs. Our results demonstrated that by shifting the location of one PNGS in A244-rgp120, and by adding two PNGS to MN-rgp120, in conjunction with the production of both proteins in a cell line that favors the incorporation of oligomannose glycans, we could significantly improve the binding by three major families of bN-mAbs. The immunogens described here represent a second generation of gp120-based vaccine immunogens that exhibit potential for use in RV144 follow-up studies.

Background. A candidate vaccine consisting of human immunodeficiency virus type 1 (HIV-1) subunit gp120 protein was found previously to be nonprotective in an efficacy trial (Vax004) despite strong antibody responses against the vaccine antigens. Here we assessed the magnitude and breadth of neutralizing antibody responses in Vax004. Methods. Neutralizing antibodies were measured against highly sensitive (tier 1) and moderately sensitive (tier 2) strains of HIV-1 subtype B in 2 independent assays. Vaccine recipients were stratified by sex, race, and high versus low behavioral risk of HIV-1 acquisition. Results. Most vaccine recipients mounted potent neutralizing antibody responses against HIV-1MN and other tier 1 viruses. Occasional weak neutralizing activity was detected against tier 2 viruses. The response against tier 1 and tier 2 viruses was significantly stronger in women than in men. Race and behavioral risk of HIV-1 acquisition had no significant effect on the response. Prior vaccination had little effect on the neutralizing antibody response that arose after infection. Conclusions. Weak overall neutralizing antibody responses against tier 2 viruses is consistent with a lack of protection in this trial. The magnitude and breadth of neutralization reported here should be useful for identifying improved vaccines.

The only evidence of vaccine-induced protection from HIV acquisition in humans was obtained in the RV144 HIV vaccine clinical trial. One immune correlate of risk in RV144 was observed to be higher titers of vaccine-induced antibodies (Abs) reacting with a 23-mer non-glycosylated peptide with the same amino acid sequence as a segment in the second variable (V2) loop of the MN strain of HIV. We used NMR to analyze the dynamic 3D structure of this peptide. Distance restraints between spatially proximate inter-residue protons were calculated from NOE cross peak intensities and used to constrain a thorough search of all possible conformations of the peptide. α-helical folding was strongly preferred by part of the peptide. A high-throughput structure prediction of this segment in all circulating HIV strains demonstrated that α-helical conformations are preferred by this segment almost universally across all subtypes. Notably, α-helical conformations of this segment of the V2 loop cluster cross-subtype-conserved amino acids on one face of the helix and the variable amino acid positions on the other in a semblance of an amphipathic α-helix. Accordingly, some Abs that protected against HIV in RV144 may have targeted a specific, conserved α-helical peptide epitope in the V2 loop of HIV's surface envelope glycoprotein.

BackgroundAn objective of the first efficacy trial of a candidate vaccine containing recombinant human immunodeficiency virus (HIV) type 1 envelope glycoprotein 120 (rgp120) antigens was to assess correlations between antibody responses to rgp120 and the incidence of HIV-1 infection MethodsWithin the randomized trial (for vaccinees, n=3598; for placebo recipients, n=1805), binding and neutralizing antibody responses to rgp120 were quantitated. A case-cohort design was used to study correlations between antibody levels and HIV-1 incidence ResultsPeak antibody levels were significantly inversely correlated with HIV-1 incidence. The relative risk (RR) of infection was 0.63 (95% confidence interval, 0.45–0.89) per log10 higher neutralization titer against HIV-1MN, and the RRs of infection for second-, third-, and fourth-quartile responses of antibody blocking of gp120 binding to soluble CD4 versus first-quartile responses (the lowest responses) were 0.35, 0.28, and 0.22, respectively ConclusionsDespite inducing a complex, robust immune response, the vaccine was unable to reduce the incidence of HIV-1. Two interpretations of the correlative results are that the levels of antibodies (i) caused both an increased (low responders) and decreased (high responders) risk of HIV-1 acquisition or (ii) represented a correlate of susceptibility to HIV-1 but had no causal effect on susceptibility. Although the data cannot definitively discriminate between these 2 explanations, (ii) appears to be more likely

A major challenge in HIV vaccine development is the identification of immunogens able to elicit broadly neutralizing antibodies (bNAbs). While remarkable progress has been made in the isolation and characterization of bNAbs, the epitopes they recognize appear to be poorly immunogenic. Thus, none of the candidate vaccines developed to date has induced satisfactory levels of neutralizing antibodies to the HIV envelope protein (Env). One approach to the problem of poor immunogenicity is to build vaccines based on envelope (env) genes retrieved from rare individuals termed elite neutralizers (ENs) who at one time possessed specific sequences that stimulated the formation of bNAbs. Env proteins selected from these individuals could possess uncommon, yet to be defined, structural features that enhance the immunogenicity of epitopes recognized by bNAbs. Here we describe the recovery of envs from an EN that developed unusually broad and potent bNAbs. As longitudinal specimens were not available, we combined plasma and provirus sequences acquired from a single time-point to infer a phylogenetic tree. Combining ancestral reconstruction data with virus neutralization data allowed us to sift through the myriad of virus quasi-species that evolved in this individual to identify envelope sequences from the nodes that appeared to define the transition from neutralization sensitive envs to the neutralization resistant envs that occur in EN plasma. Synthetic genes from these nodes were functional in infectivity assays and sensitive to neutralization by bNAbs, and may provide a novel source of immunogens for HIV vaccine development.

Recombinant gp120 (MN-rgp120) was a major component of the AIDSVAX B/E vaccine used in the RV144 trial. This was the first clinical trial to show that vaccination could prevent HIV infection in humans. A recent RV144 correlates of protection study found that protection correlated with the presence of antibodies to the V2 domain. It has been proposed that antibodies to the α4β7 binding site in the V2 domain might prevent HIV-1 infection by blocking the ability of virions to recognize α4β7 on activated T-cells. In this study we investigated the specificity of monoclonal antibodies (MAbs) to the V2 domain of MN-rgp120 and examined the possibility that these antibodies could inhibit the binding of MN-rgp120 to the α4β7 integrin. Nine MAbs to the V2 domain were isolated from mice immunized with recombinant envelope proteins. The ability of these MAbs to inhibit HIV infection, block the binding of gp120 to CD4, and block the binding of MN-rgp120 to the α4β7 integrin was measured. Mutational analysis showed that eight of the MAbs recognized two immunodominant clusters of amino acids (166-168 and 178-183) located at either end of the C strand within the four-strand anti-parallel sheet structure comprising the V1/V2 domain. These studies showed that the antigenic structure of the V2 domain is exceedingly complex and that MAbs isolated from mice immunized with MN-rgp120 exhibited a high level of strain specificity compared to MAbs to the V2 domain isolated from HIV-infected humans. We found that immunization with MN-rgp120 readily elicits antibodies to the V2 domain and some of these were able to block the binding of MN-rgp120 to the α4β7 integrin.