Kathryn A. Mesa

Range‐wide genetic homogeneity in the California sea mussel (Mytilus californianus): a comparison of allozymes, nuclear DNA markers, and mitochondrial DNA sequences

We tested for genetic differentiation among six populations of California sea mussels (Mytilus californianus) sampled across 4000 km of its geographical range by comparing patterns of variation at four independent types of genetic markers: allozymes, single‐copy nuclear DNA markers, and DNA sequences from the male and female mitochondrial genomes. Despite our extensive sampling and genotyping efforts, we detected no significant differences among localities and no signal of isolation by distance suggesting that M. californianus is genetically homogeneous throughout its range. This concordance differs from similar studies on other mytilids, especially in the role of postsettlement selection generating differences between exposed coastal and estuarine habitats. To assess if this homogeneity was due to M. californianus not inhabiting estuarine environments, we reviewed studies comparing allozymes with other classes of nuclear DNA markers. Although both types of markers gave broadly consistent results, there was a bias favouring studies in which allozymes were more divergent than DNA markers (nine to three) and a disproportionate number of these cases involved marine taxa (seven). Furthermore, allozymes were significantly more heterogeneous than DNA markers in three of the four studies that sampled coastal and estuarine habitats. We conclude that the genetic uniformity exhibited by M. californianus may result from a combination of extensive gene flow and the lack of exposure to strong selective gradients across its range.

Sequences in Glycoprotein gp41, the CD4 Binding Site, and the V2 Domain Regulate Sensitivity and Resistance of HIV-1 to Broadly Neutralizing Antibodies

ABSTRACT 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.

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

Background: Antibodies to glycan-dependent and -independent epitopes in the V1/V2 domain of HIV-1 gp120 have been associated with protective immunity. Results: Novel gp120s and scaffolds able to target antibodies to both types of epitopes in the V1/V2 domain were developed. Conclusion: Improved antibody responses to the V1/V2 domain were observed. Significance: The immunogens described may provide improved HIV-1 vaccines. 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 167–171) adjacent to the junction of the B and C strands of the four-stranded V1/V2 domain β-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.

Glycans flanking the hypervariable connecting peptide between the A and B strands of the V1/V2 domain of HIV-1 gp120 confer resistance to antibodies that neutralize CRF01_AE viruses.

Understanding the molecular determinants of sensitivity and resistance to neutralizing antibodies is critical for the development of vaccines designed to prevent HIV infection. In this study, we used a genetic approach to characterize naturally occurring polymorphisms in the HIV envelope protein that conferred neutralization sensitivity or resistance. Libraries of closely related envelope genes, derived from virus quasi-species, were constructed from individuals infected with CRF01_AE viruses. The libraries were screened with plasma containing broadly neutralizing antibodies, and neutralization sensitive and resistant variants were selected for sequence analysis. In vitro mutagenesis allowed us to identify single amino acid changes in three individuals that conferred resistance to neutralization by these antibodies. All three mutations created N-linked glycosylation sites (two at N136 and one at N149) proximal to the hypervariable connecting peptide between the C-terminus of the A strand and the N-terminus of the B strand in the four-stranded V1/V2 domain β-sheet structure. Although N136 has previously been implicated in the binding of broadly neutralizing monoclonal antibodies, this glycosylation site appears to inhibit the binding of neutralizing antibodies in plasma from HIV-1 infected subjects. Previous studies have reported that the length of the V1/V2 domain in transmitted founder viruses is shorter and possesses fewer glycosylation sites compared to viruses isolated from chronic infections. Our results suggest that vaccine immunogens based on recombinant envelope proteins from clade CRF01_AE viruses might be improved by inclusion of envelope proteins that lack these glycosylation sites. This strategy might improve the efficacy of the vaccines used in the partially successful RV144 HIV vaccine trial, where the two CRF01_AE immunogens (derived from the A244 and TH023 isolates) both possessed glycosylation sites at N136 and N149.

Fragments of the V1/V2 domain of HIV-1 glycoprotein 120 engineered for improved binding to the broadly neutralizing PG9 antibody.

The V1/V2 domain of the HIV-1 envelope protein gp120 possesses two important epitopes: a glycan-dependent epitope recognized by the prototypic broadly neutralizing monoclonal antibody (bN-mAb), PG9, as well as an epitope recognized by non-neutralizing antibodies that has been associated with protection from HIV infection in the RV144 HIV vaccine trial. Because both of these epitopes are poorly immunogenic in the context of full length envelope proteins, immunization with properly folded and glycosylated fragments (scaffolds) represents a potential way to enhance the immune response to these specific epitopes. Previous studies showed that V1/V2 domain scaffolds could be produced from a few selected isolates, but not from many of the isolates that would be advantageous in a multivalent vaccine. In this paper, we used a protein engineering approach to improve the conformational stability and antibody binding activity of V1/V2 domain scaffolds from multiple diverse isolates, including several that were initially unable to bind the prototypic PG9 bN-mAb. Significantly, this effort required replicating both the correct glycan structure as well as the β-sheet structure required for PG9 binding. Although scaffolds incorporating the glycans required for PG9 binding (e.g., mannose-5) can be produced using glycosylation inhibitors (e.g., swainsonine), or mutant cell lines (e.g. GnTI(-) 293 HEK), these are not practical for biopharmaceutical production of proteins intended for clinical trials. In this report, we describe engineered glycopeptide scaffolds from three different clades of HIV-1 that bind PG9 with high affinity when expressed in a wildtype cell line suitable for biopharmaceutical production. The mutations that improved PG9 binding to scaffolds produced in normal cells included amino acid positions outside of the antibody contact region designed to stabilize the β-sheet and turn structures. The scaffolds produced address three major problems in HIV vaccine development: (1) improving antibody responses to poorly immunogenic epitopes in the V1/V2 domain; (2) eliminating antibody responses to highly immunogenic (decoy) epitopes outside the V1/V2 domain; and (3) enabling the production of V1/V2 scaffolds in a cell line suitable for biopharmaceutical production.

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.

Characterization of a monoclonal antibody to a novel glycan-dependent epitope in the V1/V2 domain of the HIV-1 envelope protein, gp120.

Abstract Recent studies have described several broadly neutralizing monoclonal antibodies (bN-mAbs) that recognize glycan-dependent epitopes (GDEs) in the HIV-1 envelope protein, gp120. These were recovered from HIV-1 infected subjects, and several (e.g., PG9, PG16, CH01, CH03) target glycans in the first and second variable (V1/V2) domain of gp120. The V1/V2 domain is thought to play an important role in conformational masking, and antibodies to the V1/V2 domain were recently identified as the only immune response that correlated with protection in the RV144 HIV-1 vaccine trial. While the importance of antibodies to polymeric glycans is well established for vaccines targeting bacterial diseases, the importance of antibodies to glycans in vaccines targeting HIV has only recently been recognized. Antibodies to GDEs may be particularly significant in HIV vaccines based on gp120, where 50% of the molecular mass of the envelope protein is contributed by N-linked carbohydrate. However, few studies have reported antibodies to GDEs in humans or animals immunized with candidate HIV-1 vaccines. In this report, we describe the isolation of a mouse mAb, 4B6, after immunization with the extracellular domain of the HIV-1 envelope protein, gp140. Epitope mapping using glycopeptide fragments and in vitro mutagenesis showed that binding of this antibody depends on N-linked glycosylation at asparagine N130 (HXB2 numbering) in the gp120 V1/V2 domain. Our results demonstrate that, in addition to natural HIV-1 infection, immunization with recombinant proteins can elicit antibodies to the GDEs in the V1/V2 domain of gp120. Although little is known regarding conditions that favor antibody responses to GDEs, our studies demonstrate that these antibodies can arise from a short-term immunization regimen. Our results suggest that antibodies to GDEs are more common than previously suspected, and that further analysis of antibody responses to the HIV-1 envelope protein will lead to the discovery of additional antibodies to GDEs.

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.

Development of a Stable MGAT1− CHO Cell Line to Produce Clade C gp120 With Improved Binding to Broadly Neutralizing Antibodies

The high rate of new HIV infections, particularly in Sub-Saharan Africa, emphasizes the need for a safe and effective vaccine to prevent acquired immunodeficiency syndrome (AIDS). To date, the only HIV vaccine trial that has exhibited protective efficacy in humans was the RV144 study completed in Thailand. The finding that protection correlated with antibodies to gp120 suggested that increasing the quality or magnitude of the antibody response that recognize gp120 might improve the modest yet significant protection (31.2%) achieved with this immunization regimen. However, the large-scale production of rgp120 suitable for clinical trials has been challenging due, in part, to low productivity and difficulties in purification. Moreover, the antigens that are currently available were produced largely by the same technology used in the early 1990s and fail to incorporate unique carbohydrates presented on HIV virions required for the binding of several major families of broadly neutralizing antibodies (bNAbs). Here we describe the development of a high-yielding CHO cell line expressing rgp120 from a clade C isolate (TZ97008), representative of the predominant circulating HIV subtype in Southern Africa and Southeast Asia. This cell line, produced using robotic selection, expresses high levels (1.2 g/L) of the TZ97008 rgp120 antigen that incorporates oligomannose glycans required for binding to multiple glycan dependent bNAbs. The resulting rgp120 displays a lower degree of net charge and glycoform heterogeneity as compared to rgp120s produced in normal CHO cells. This homogeneity in net charge facilitates purification by filtration and ion exchange chromatography methods, eliminating the need for expensive custom-made lectin, or immunoaffinity columns. The results described herein document the availability of a novel cell line for the large-scale production of clade C gp120 for clinical trials. Finally, the strategy used to produce a TZ97008 gp120 in the MGAT− CHO cell line can be applied to the production of other candidate HIV vaccines.

Mapping epitopes on CRF01_AE viruses recognized by broadly neutralizing antibodies in sera from elite neutralizers from North America and Thailand.

Background The RV144 trial has rekindled interest in defining the epitopes responsible for the neutralization of CRF01_AE (clade E) viruses. In this study we used a novel method, swarm analysis, to map epitopes on Thai viruses recognized by broadly neutralizing (bN) antibodies in HIV+ sera. Swarm analysis relies on the swarm of closely related quasi-species that evolve in each HIV+ individual. In these studies we analyzed the bN activity in sera from elite neutralizers (ENs) against Thai viruses.