Immune response to SARS-CoV-2 variants after immunization with different vaccines in Mexico.

There is limited information on the antibody responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in subjects from developing countries with populations having a high incidence of co-morbidities. Here, we analysed the immunogenicity of homologous schemes using the ChAdOx1-S, Sputnik V, or BNT162b2 vaccines and the effect of a booster dose with ChAdOx1-S in middle-aged adults who were seropositive or seronegative to the SARS-CoV-2 spike protein before vaccination. The study was conducted post-vaccination with a follow-up of 4 months for antibody titre using enzyme-linked immunosorbent assay (ELISA) and pseudovirus (PV) neutralization assays (PNAs). All three vaccines elicited a superior IgG anti-receptor-binding domain (RBD) and neutralization response against the Alpha and Delta variants when administered to individuals with a previous infection by SARS-CoV-2. The booster dose spiked the neutralization activity among individuals with and without a prior SARS-CoV-2 infection. The ChAdOx1-S vaccine induced weaker antibody responses in infection-naive subjects. A follow-up of 4 months post-vaccination showed a drop in antibody titre, with about 20% of the infection-naive and 100% of SARS-CoV-2 pre-exposed participants with detectable neutralization capacity against Alpha pseudovirus (Alpha-PV) and Delta PV (Delta-PV). Our observations support the use of different vaccines in a country with high seroprevalence at the vaccination time.

Structural insights into the broad protection against H1 influenza viruses by a computationally optimized hemagglutinin vaccine.

Influenza virus poses an ongoing human health threat with pandemic potential. Due to mutations in circulating strains, formulating effective vaccines remains a challenge. The use of computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) proteins is a promising vaccine strategy to protect against a wide range of current and future influenza viruses. Though effective in preclinical studies, the mechanistic basis driving the broad reactivity of COBRA proteins remains to be elucidated. Here, we report the crystal structure of the COBRA HA termed P1 and identify antigenic and glycosylation properties that contribute to its immunogenicity. We further report the cryo-EM structure of the P1-elicited broadly neutralizing antibody 1F8 bound to COBRA P1, revealing 1F8 to recognize an atypical receptor binding site epitope via an unexpected mode of binding.

Structure-Based Design and Antigenic Validation of Respiratory Syncytial Virus G Immunogens.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease of children, the elderly, and immunocompromised individuals. Currently, there are no FDA-approved RSV vaccines. The RSV G glycoprotein is used for viral attachment to host cells and impairment of host immunity by interacting with the human chemokine receptor CX3CR1. Antibodies that disrupt this interaction are protective against infection and disease. Nevertheless, development of an RSV G vaccine antigen has been hindered by its low immunogenicity and safety concerns. A previous study described three engineered RSV G proteins containing single-point mutations that induce higher levels of IgG antibodies and have improved safety profiles compared to wild-type RSV G (H. C. Bergeron, J. Murray, A. M. Nuñez Castrejon, et al., Viruses 13:352, 2021, https://doi.org/10.3390/v13020352). However, it is unclear if the mutations affect RSV G protein folding and display of its conformational epitopes. In this study, we show that the RSV G S177Q protein retains high-affinity binding to protective human and mouse monoclonal antibodies and has equal reactivity as wild-type RSV G protein to human reference immunoglobulin to RSV. Additionally, we determined the high-resolution crystal structure of RSV G S177Q protein in complex with the anti-RSV G antibody 3G12, further validating its antigenic structure. These studies show for the first time that an engineered RSV G protein with increased immunogenicity and safety retains conformational epitopes to high-affinity protective antibodies, supporting its further development as an RSV vaccine immunogen. IMPORTANCE Respiratory syncytial virus (RSV) causes severe lower respiratory diseases of children, the elderly, and immunocompromised populations. There currently are no FDA-approved RSV vaccines. Most vaccine development efforts have focused on the RSV F protein, and the field has generally overlooked the receptor-binding antigen RSV G due to its poor immunogenicity and safety concerns. However, single-point mutant RSV G proteins have been previously identified that have increased immunogenicity and safety. In this study, we investigate the antibody reactivities of three known RSV G mutant proteins. We show that one mutant RSV G protein retains high-affinity binding to protective monoclonal antibodies, is equally recognized by anti-RSV antibodies in human sera, and forms the same three-dimensional structure as the wild-type RSV G protein. Our study validates the structure-guided design of the RSV G protein as an RSV vaccine antigen.

Targeting the Conserved Stem Loop 2 Motif in the SARS-CoV-2 Genome.

RNA structural elements occur in numerous single-stranded positive-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3' untranslated region (UTR) in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts imply a key role for s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodeled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 replication in cell culture. Our results thus permit us to suggest that the s2m element is readily targeted by ASOs, which show promise as antiviral agents. IMPORTANCE The highly conserved stem-loop 2 motif (s2m) is found in the genomes of many RNA viruses, including SARS-CoV-2. Our findings indicate that the s2m element can be targeted by antisense oligonucleotides. The antiviral potential of this element represents a promising start for further research into targeting conserved elements in RNA viruses.

Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry.

Serological testing to evaluate antigen-specific antibodies in plasma is generally performed by rapid lateral flow test strips that lack quantitative results or by high complexity immunoassays that are time- and labor-intensive but provide semi-quantitative results. Here, we describe a novel application of biolayer interferometry for the rapid detection of antigen-specific antibody levels in plasma samples, and demonstrate its utility for quantification of SARS-CoV-2 antibodies. Our biolayer interferometry immunosorbent assay (BLI-ISA) utilizes single-use biosensors in an automated "dip-and-read" format, providing real-time optical measurements of antigen loading, plasma antibody binding, and antibody isotype detection. Complete semi-quantitative results are obtained in less than 20 min. BLI-ISA meets or exceeds the performance of high complexity methods such as Enzyme-Linked Immunosorbent Assay (ELISA) and Chemiluminescent Immunoassay. Importantly, our method can be immediately implemented on existing BLI platforms for urgent COVID-19 studies, such as serosurveillance and the evaluation of vaccine candidates. In a broader sense, BLI-ISA can be developed as a novel diagnostic platform to evaluate antibodies and other biomolecules in clinical specimens.

Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry.

Serological testing to evaluate antigen-specific antibodies in plasma is generally performed by rapid lateral flow test strips that lack quantitative results or by high complexity immunoassays that are time- and labor-intensive but provide quantitative results. Here, we describe a novel application of biolayer interferometry for the rapid detection of antigen-specific antibody levels in plasma samples, and demonstrate its utility for quantification of SARS-CoV-2 antibodies. Our biolayer interferometry immunosorbent assay (BLI-ISA) utilizes single-use biosensors in an automated "dip-and-read" format, providing real-time optical measurements of antigen loading, plasma antibody binding, and antibody isotype detection. Complete quantitative results are obtained in less than 20 minutes. BLI-ISA meets or exceeds the performance of high complexity methods such as Enzyme-Linked Immunosorbent Assay (ELISA) and Chemiluminescent Immunoassay. Importantly, our method can be immediately implemented on existing BLI platforms for urgent COVID-19 studies, such as serosurveillance and the evaluation of vaccine candidates. In a broader sense, BLI-ISA can be developed as a novel diagnostic platform to evaluate antibodies and other biomolecules in clinical specimens.

Unusual Cysteine Content in V1 Region of gp120 From an Elite Suppressor That Produces Broadly Neutralizing Antibodies.

Although it is now possible to produce recombinant HIV envelope glycoproteins (Envs) with epitopes recognized by the 5-6 major classes of broadly neutralizing antibodies (bNAbs), these have failed to consistently stimulate the formation of bNAbs in immunized animals or humans. In an effort to identify new immunogens better able to elicit bNAbs, we are studying Envs derived from rare individuals who possess bNAbs and are able to control their infection without the need for anti-retroviral drugs (elite supressors or ES), hypothesizing that in at least some people the antibodies may mediate durable virus control. Because virus evolution in people with the ES only phenotype was reported to be limited, we reasoned the Env proteins recovered from these individuals may more closely resemble the Envs that gave rise to bNAbs compared to the highly diverse viruses isolated from normal progressors. Using a phenotypic assay, we screened 25 controllers and identified two for more detailed investigation. In this study, we examined 20 clade B proviral sequences isolated from an African American woman, who had the rare bNAb/ES phenotype. Phylogenetic analysis of proviral envelope sequences demonstrated low genetic diversity. Envelope proteins were unusual in that most possessed two extra cysteines within an elongated V1 region. In this report, we examine the impact of the extra cysteines on the binding to bNAbs, virus infectivity, and sensitivity to neutralization. These data suggest structural motifs in V1 can affect infectivity, and that rare viruses may be prevented from developing escape.

Ancestral sequences from an elite neutralizer proximal to the development of neutralization resistance as a potential source of HIV vaccine immunogens.

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.

Production of soluble pMHC-I molecules in mammalian cells using the molecular chaperone TAPBPR.

Current approaches for generating major histocompatibility complex (MHC) Class-I proteins with desired bound peptides (pMHC-I) for research, diagnostic and therapeutic applications are limited by the inherent instability of empty MHC-I molecules. Using the properties of the chaperone TAP-binding protein related (TAPBPR), we have developed a robust method to produce soluble, peptide-receptive MHC-I molecules in Chinese Hamster Ovary cells at high yield, completely bypassing the requirement for laborious refolding from inclusion bodies expressed in E.coli. Purified MHC-I/TAPBPR complexes can be prepared for multiple human allotypes, and exhibit complex glycan modifications at the conserved Asn 86 residue. As a proof of concept, we demonstrate both HLA allele-specific peptide binding and MHC-restricted antigen recognition by T cells for two relevant tumor-associated antigens. Our system provides a facile, high-throughput approach for generating pMHC-I antigens to probe and expand TCR specificities present in polyclonal T cell repertoires.

Production of a recombinant monoclonal antibody to Herpes Simplex Virus glycoprotein D for immunoaffinity purification of tagged proteins.

We have developed a stable Chinese Hamster Ovary (CHO) cell line for the production of a recombinant monoclonal antibody (mAb) to a short protein sequence derived from the N-terminus of human herpes simplex virus type 1 glycoprotein D (HSV-1 gD). The antibody (designated r34.1) provides a useful tool for the immunoaffinity purification of HSV-1 gD tagged proteins, and provides a generic purification system by which various proteins and peptides can be purified. Recombinant 34.1 was assembled using cDNA derived from a HSV-1 gD specific murine hybridoma engineered to encode a full-length IgG molecule. Antibody expression cassettes were transfected into CHO-S cells, and a stable cell-line expressing up to 500 mg/L of antibody, isolated. Affinity purified r34.1 exhibited nanomolar affinity for its cognate ligand, and is stable throughout multiple cycles of immunoaffinity purification involving ligand binding at neutral pH, followed by acid elution. The HSV-1 gD tag expression and purification strategy has been used to enhance the secretion and purification of several vaccine immunogens including HIV envelope protein rgp120s, but the protocol has potential for generic application.

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.

Structural Simplicity and Mechanistic Complexity in the Hammerhead Ribozyme.

Natural or full-length hammerhead ribozymes are up to 1000-fold more active than their minimal counterparts that lack a complex tertiary interaction that pre-organizes and stabilizes the ribozyme active site, positioning RNA functional groups to facilitate acid-base catalysis. The recent discovery that a single tertiary contact (an AU Hoogsteen pair) between Stems I and II confers essentially all of the enhanced activity greatly simplifies our understanding of the structural requirements for hammerhead ribozyme activity. In contrast, the simplest mechanistic interpretations are challenged with the presentation of more complex alternatives. These alternatives are elucidated and critically analyzed in the context of several of the active hammerhead ribozyme structures now available.

CRISPR/Cas9 gene editing for the creation of an MGAT1-deficient CHO cell line to control HIV-1 vaccine glycosylation.

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.

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.

Minimal Hammerhead Ribozymes with Uncompromised Catalytic Activity.

We report here that a single additional trans-Hoogsteen base-pairing interaction in the minimal hammerhead ribozyme transforms an RNA sequence possessing typically modest catalytic activity into one possessing greatly enhanced catalytic activity that is instead typical of full-length natural hammerhead RNAs that have additional extensive tertiary contact interactions. Formation of this additional base-pairing interaction requires only that the substrate RNA sequence contains a U at a position seven nucleotides 3' to the cleavage site. No additions or changes are required in the minimal hammerhead ribozyme enzyme strand sequence (providing that the naturally occurring GUGA tetraloop of Stem II is maintained). This finding unambiguously demonstrates that a single Hoogsteen base-pairing interaction, in full-length hammerheads possessing this interaction, is sufficient for stabilizing the ribozyme active site, including alignment of the attacking nucleophile for the required inline hammerhead ribozyme reaction mechanism. This finding also implies that the idiosyncratic arrays of additional tertiary contacts observed in all naturally occurring full-length hammerhead sequences have evolved to prevent deleterious alternative pairing interactions within the context of the variety of natural sequences arising in vivo. Finally, this finding greatly simplifies and rationalizes the design of fast-cleaving engineered synthetic ribozymes as RNA nucleolytic reagents and as subjects for enzyme kinetics and mechanistic investigations.

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 ß-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.

RNA catalysis, thermodynamics and the origin of life.

The RNA World Hypothesis posits that the first self-replicating molecules were RNAs. RNA self-replicases are, in general, assumed to have employed nucleotide 5'-polyphosphates (or their analogues) as substrates for RNA polymerization. The mechanism by which these substrates might be synthesized with sufficient abundance to supply a growing and evolving population of RNAs is problematic for evolutionary hypotheses because non-enzymatic synthesis and assembly of nucleotide 5'-triphosphates (or other analogously activated phosphodiester species) is inherently difficult. However, nucleotide 2',3'-cyclic phosphates are also phosphodiesters, and are the natural and abundant products of RNA degradation. These have previously been dismissed as viable substrates for prebiotic RNA synthesis. We propose that the arguments for their dismissal are based on a flawed assumption, and that nucleotide 2',3'-cyclic phosphates in fact possess several significant, advantageous properties that indeed make them particularly viable substrates for prebiotic RNA synthesis. An RNA World hypothesis based upon the polymerization of nucleotide 2',3'-cyclic phosphates possesses additional explanatory power in that it accounts for the observed ribozyme "fossil record", suggests a viable mechanism for substrate transport across lipid vesicle boundaries of primordial proto-cells, circumvents the problems of substrate scarcity and implausible synthetic pathways, provides for a primitive but effective RNA replicase editing mechanism, and definitively explains why RNA, rather than DNA, must have been the original catalyst. Finally, our analysis compels us to propose that a fundamental and universal property that drives the evolution of living systems, as well as pre-biotic replicating molecules (be they composed of RNA or protein), is that they exploit chemical reactions that already possess competing kinetically-preferred and thermodynamically-preferred pathways in a manner that optimizes the balance between the two types of pathways.

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

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.

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.