Robotic selection for the rapid development of stable CHO cell lines for HIV vaccine production.

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.

Glycan modifications to the gp120 immunogens used in the RV144 vaccine trial improve binding to broadly neutralizing antibodies.

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.

Effects of Cross-Presentation, Antigen Processing, and Peptide Binding in HIV Evasion of T Cell Immunity.

Unlike cytosolic processing and presentation of viral Ags by virus-infected cells, Ags first expressed in infected nonprofessional APCs, such as CD4+ T cells in the case of HIV, are taken up by dendritic cells and cross-presented. This generally requires entry through the endocytic pathway, where endosomal proteases have first access for processing. Thus, understanding virus escape during cross-presentation requires an understanding of resistance to endosomal proteases, such as cathepsin S (CatS). We have modified HIV-1MN gp120 by mutating a key CatS cleavage site (Thr322Thr323) in the V3 loop of the immunodominant epitope IGPGRAFYTT to IGPGRAFYVV to prevent digestion. We found this mutation to facilitate cross-presentation and provide evidence from MHC binding and X-ray crystallographic structural studies that this results from preservation of the epitope rather than an increased epitope affinity for the MHC class I molecule. In contrast, when the protein is expressed by a vaccinia virus in the cytosol, the wild-type protein is immunogenic without this mutation. These proof-of-concept results show that a virus like HIV, infecting predominantly nonprofessional presenting cells, can escape T cell recognition by incorporating a CatS cleavage site that leads to destruction of an immunodominant epitope when the Ag undergoes endosomal cross-presentation.

HIV-1 envelope proteins and V1/V2 domain scaffolds with mannose-5 to improve the magnitude and quality of protective antibody responses to HIV-1.

Two lines of investigation have highlighted the importance of antibodies to the V1/V2 domain of gp120 in providing protection from HIV-1 infection. First, the recent RV144 HIV-1 vaccine trial documented a correlation between non-neutralizing antibodies to the V2 domain and protection. Second, multiple broadly neutralizing monoclonal antibodies to the V1/V2 domain (e.g. PG9) have been isolated from rare infected individuals, termed elite neutralizers. Interestingly, the binding of both types of antibodies appears to depend on the same cluster of amino acids (positions 167­171) adjacent to the junction of the B and C strands of the four-stranded V1/V2 domain ß-sheet structure. However, the broadly neutralizing mAb, PG9, additionally depends on mannose-5 glycans at positions 156 and 160 for binding. Because the gp120 vaccine immunogens used in previous HIV-1 vaccine trials were enriched for complex sialic acid-containing glycans, and lacked the high mannose structures required for the binding of PG9-like mAbs, we wondered if these immunogens could be improved by limiting glycosylation to mannose-5 glycans. Here, we describe the PG9 binding activity of monomeric gp120s from multiple strains of HIV-1 produced with mannose-5 glycans. We also describe the properties of glycopeptide scaffolds from the V1/V2 domain also expressed with mannose-5 glycans. The V1/V2 scaffold from the A244 isolate was able to bind the PG9, CH01, and CH03 mAbs with high affinity provided that the proper glycans were present. We further show that immunization with A244 V1/V2 fragments alone, or in a prime/boost regimen with gp120, enhanced the antibody response to sequences in the V1/V2 domain associated with protection in the RV144 trial.

Sequences in glycoprotein gp41, the CD4 binding site, and the V2 domain regulate sensitivity and resistance of HIV-1 to broadly neutralizing antibodies.

The swarm of quasispecies that evolves in each HIV-1-infected individual represents a source of closely related Env protein variants that can be used to explore various aspects of HIV-1 biology. In this study, we made use of these variants to identify mutations that confer sensitivity and resistance to the broadly neutralizing antibodies found in the sera of selected HIV-1-infected individuals. For these studies, libraries of Env proteins were cloned from infected subjects and screened for infectivity and neutralization sensitivity. The nucleotide sequences of the Env proteins were then compared for pairs of neutralization-sensitive and -resistant viruses. In vitro mutagenesis was used to identify the specific amino acids responsible for the neutralization phenotype. All of the mutations altering neutralization sensitivity/resistance appeared to induce conformational changes that simultaneously enhanced the exposure of two or more epitopes located in different regions of gp160. These mutations appeared to occur at unique positions required to maintain the quaternary structure of the gp160 trimer, as well as conformational masking of epitopes targeted by neutralizing antibodies. Our results show that sequences in gp41, the CD4 binding site, and the V2 domain all have the ability to act as global regulators of neutralization sensitivity. Our results also suggest that neutralization assays designed to support the development of vaccines and therapeutics targeting the HIV-1 Env protein should consider virus variation within individuals as well as virus variation between individuals.

Glycoform and net charge heterogeneity in gp120 immunogens used in HIV vaccine trials.

BACKGROUND: The RV144 clinical trial showed for the first time that vaccination could provide modest but significant protection from HIV-1 infection. To understand the protective response, and to improve upon the vaccine's efficacy, it is important to define the structure of the immunogens used in the prime/boost regimen. Here we examined the heterogeneity in net charge, attributable to glycoform variation, of the gp120 immunogens contained in the AIDSVAX B/E vaccine. METHODOLOGY/PRINCIPAL FINDINGS: Isoelectric focusing and glycosidase digestion were used to assess variation in net charge of the gp120s contained in the AIDSVAX B/E vaccine used in the RV144 trial. We observed 16 variants of MN-rgp120 and 24 variants of A244-rgp120. Glycoform variation in gp120 produced in Chinese hamster ovary cells was compared to glycoform variation in gp120 produced in the 293F human embryonic kidney cell line, often used for neutralization assays. We found that gp120 variants produced in CHO cells were distinctly more acidic than gp120 variants produced in 293 cells. The effect of glycoform heterogeneity on antigenicity was assessed using monoclonal antibodies. The broadly neutralizing PG9 MAb bound to A244-rgp120, but not to MN-rgp120, whether produced in CHO or in 293. However, PG9 was able to bind with high affinity to MN-rgp120 and A244-rgp120 produced in 293 cells deficient in N-acetylglucosaminyltransferase I. CONCLUSIONS/SIGNIFICANCE: MN- and A244-rgp120 used in the RV144 trial exhibited extensive heterogeneity in net charge due to variation in sialic acid-containing glycoforms. These differences were cell line-dependent, affected the antigenicity of recombinant envelope proteins, and may affect assays used to measure neutralization. These studies, together with recent reports documenting broadly neutralizing antibodies directed against carbohydrate epitopes of gp120, suggest that glycoform variation is a key variable to be considered in the production and evaluation of subunit vaccines designed to prevent HIV infection.

Adeno-associated virus (AAV) capsid genes isolated from rat and mouse liver genomic DNA define two new AAV species distantly related to AAV-5.

Using polymerase chain reactions and genome walking strategies, adeno-associated virus (AAV)-like capsid genes were isolated from rat and mouse liver genomic DNA, where they are present at <5 copies per cell. These genes define two new species of AAVs since their amino acid sequences are <60% identical to each other or to any other AAV capsid. They are most similar to the AAV-5 and goat AAV capsids. A recombinant vector with the mouse AAV capsid and a lacZ transgene (rAAV-mo.1 lacZ) was able to transduce rodent cell lines in vitro. However, it was not able to transduce eight human cell lines or primary human fibroblasts in vitro. It did not bind heparin and its ability to transduce cells in vitro was not inhibited by heparin, mucin, or sialic acid suggesting it uses a novel entry receptor. rAAV-mo.1 lacZ was 29 times more resistant to in vitro neutralization by pooled, purified human IgG than AAV-2. In vivo, rAAV-mo.1 lacZ efficiently transduced murine ocular cells after a subretinal injection. Intramuscular injection of a rAAV-mo.1 human factor IX (hFIX) vector into mice resulted in no detectable hFIX in plasma, but intravenous injection resulted in high plasma levels of hFIX, equivalent to that obtained from a rAAV-8 hFIX vector. Biodistribution analysis showed that rAAV-mo.1 primarily transduced liver after an intravenous injection. These AAV capsids may be useful for gene transfer in rodents.

Mutations on the external surfaces of adeno-associated virus type 2 capsids that affect transduction and neutralization.

Mutations were made at 64 positions on the external surface of the adeno-associated virus type 2 (AAV-2) capsid in regions expected to bind antibodies. The 127 mutations included 57 single alanine substitutions, 41 single nonalanine substitutions, 27 multiple mutations, and 2 insertions. Mutants were assayed for capsid synthesis, heparin binding, in vitro transduction, and binding and neutralization by murine monoclonal and human polyclonal antibodies. All mutants made capsid proteins within a level about 20-fold of that made by the wild type. All but seven mutants bound heparin as well as the wild type. Forty-two mutants transduced human cells at least as well as the wild type, and 10 mutants increased transducing activity up to ninefold more than the wild type. Eighteen adjacent alanine substitutions diminished transduction from 10- to 100,000-fold but had no effect on heparin binding and define an area (dead zone) required for transduction that is distinct from the previously characterized heparin receptor binding site. Mutations that reduced binding and neutralization by a murine monoclonal antibody (A20) were localized, while mutations that reduced neutralization by individual human sera or by pooled human, intravenous immunoglobulin G (IVIG) were dispersed over a larger area. Mutations that reduced binding by A20 also reduced neutralization. However, a mutation that reduced the binding of IVIG by 90% did not reduce neutralization, and mutations that reduced neutralization by IVIG did not reduce its binding. Combinations of mutations did not significantly increase transduction or resistance to neutralization by IVIG. These mutations define areas on the surface of the AAV-2 capsid that are important determinants of transduction and antibody neutralization.