Membrane Env Liposomes Facilitate Immunization with Multivalent Full-Length HIV Spikes.

A major goal of HIV vaccine design is to elicit broadly neutralizing antibodies (bNAbs). Such bNAbs target HIV's trimeric, membrane-embedded envelope glycoprotein spikes (mEnv). Soluble Env (sEnv) trimers have been used as vaccines, but engineering sEnvs for stability, multivalency, and desired antigenicity is problematic and deletes key neutralizing epitopes on glycoprotein 41 (gp41) while creating neoepitopes that elicit unwanted antibodies. Meanwhile, multivalent mEnv vaccines are challenging to develop due to trimer instability and low mEnv copy number amid other extraneous proteins on virus-like particles. Here, we describe a multivalent mEnv vaccine platform that does not require protein engineering or extraneous proteins. mEnv trimers were fixed, purified, and combined with naked liposomes in mild detergent. On removal of detergent, mEnv spikes were observed embedded in liposome particles (mean diameter, 133 nm) in correct orientation. These particles were recognized by HIV bNAbs and not non-NAbs and are designated mEnv liposomes (MELs). Following a sequential immunization scheme in rabbits, MELs elicited antibodies that neutralized tier 2 HIV isolates. Analysis of serum antibody specificities, including those to epitopes involving a missing conserved N-glycosylation site at position 197 near the CD4 binding site on two of the immunogens, provides clues on how NAb responses can be improved with modified immunogens. In sum, MELs are a biochemically defined platform that enables rational immunization strategies to elicit HIV bNAbs using multimerized mEnv. IMPORTANCE A vaccine that induced broadly neutralizing antibodies against HIV would likely end the AIDS pandemic. Such antibodies target membrane-embedded envelope glycoprotein spikes (mEnv) that HIV uses to enter cells. Due to HIV Env's low expression and instability, soluble stabilized Env trimers have been used as vaccine candidates, but these have an altered base that disrupts targets of HIV broadly neutralizing antibodies that bind near the membrane and are not available for all HIV isolates. Here, we describe membrane Env liposomes (MELs) that display a multivalent array of stable mEnvs on liposome particles. MELs showed the expected antibody recognition properties, including targeting parts of mEnv missing on soluble Envs. Immunization with MELs elicited antibodies that neutralized diverse HIV isolates. The MEL platform facilitates vaccine development with potentially any HIV Env at high valency, and a similar approach may be useful for eliciting antibodies to membrane-embedded targets of therapeutic interest.

Covalent Linkage of HIV-1 Trimers to Synthetic Liposomes Elicits Improved B Cell and Antibody Responses.

We have demonstrated that a liposomal array of well-ordered trimers enhances B cell activation, germinal center formation, and the elicitation of tier-2 autologous neutralizing antibodies. Previously, we coupled well-ordered cleavage-independent NFL trimers via their C-terminal polyhistidine tails to nickel lipids integrated into the lipid bilayer. Despite favorable in vivo effects, concern remained over the potentially longer-term in vivo instability of noncovalent linkage of the trimers to the liposomes. Accordingly, we tested both cobalt coupling and covalent linkage of the trimers to the liposomes by reengineering the polyhistidine tail to include a free cysteine on each protomer of model BG505 NFL trimers to allow covalent linkage. Both cobalt and cysteine coupling resulted in a high-density array of NFL trimers that was stable in both 20% mouse serum and 100 mM EDTA, whereas the nickel-conjugated trimers were not stable under these conditions. Binding analysis and calcium flux with anti-Env-specific B cells confirmed that the trimers maintained conformational integrity following coupling. Following immunization of mice, serologic analysis demonstrated that the covalently coupled trimers elicited Env-directed antibodies in a manner statistically significantly improved compared to soluble trimers and nickel-conjugated trimers. Importantly, the covalent coupling not only enhanced gp120-directed responses compared to soluble trimers, it also completely eliminated antibodies directed to the C-terminal His tag located at the "bottom" of the spike. In contrast, soluble and noncovalent formats efficiently elicited anti-His tag antibodies. These data indicate that covalent linkage of well-ordered trimers to liposomes in high-density array displays multiple advantages in vitro and in vivoIMPORTANCE Enveloped viruses typically encode a surface-bound glycoprotein that mediates viral entry into host cells and is a primary target for vaccine design. Liposomes with modified lipid head groups have a unique feature of capturing and displaying antigens on their surfaces, mimicking the native pathogens. Our first-generation nickel-based liposomes captured HIV-1 Env glycoprotein trimers via a noncovalent linkage with improved efficacy over soluble glycoprotein in activating germinal center B cells and eliciting tier-2 autologous neutralizing antibodies. In this study, we report the development of second-generation cobalt- and maleimide-based liposomes that have improved in vitro stability over nickel-based liposomes. In particular, the maleimide liposomes captured HIV-1 Env trimers via a more stable covalent bond, resulting in enhanced germinal center B cell responses that generated higher antibody titers than the soluble trimers and liposome-bearing trimers via noncovalent linkages. We further demonstrate that covalent coupling prevents release of the trimers prior to recognition by B cells and masks a nonneutralizing determinant located at the bottom of the trimer.

Dense Array of Spikes on HIV-1 Virion Particles.

HIV-1 is rare among viruses for having a low number of envelope glycoprotein (Env) spikes per virion, i.e., ∼7 to 14. This exceptional feature has been associated with avoidance of humoral immunity, i.e., B cell activation and antibody neutralization. Virus-like particles (VLPs) with increased density of Env are being pursued for vaccine development; however, these typically require protein engineering that alters Env structure. Here, we used instead a strategy that targets the producer cell. We employed fluorescence-activated cell sorting (FACS) to sort for cells that are recognized by trimer cross-reactive broadly neutralizing antibody (bnAb) and not by nonneutralizing antibodies. Following multiple iterations of FACS, cells and progeny virions were shown to display higher levels of antigenically correct Env in a manner that correlated between cells and cognate virions (P = 0.027). High-Env VLPs, or hVLPs, were shown to be monodisperse and to display more than a 10-fold increase in spikes per particle by electron microscopy (average, 127 spikes; range, 90 to 214 spikes). Sequencing revealed a partial truncation in the C-terminal tail of Env that had emerged in the sort; however, iterative rounds of "cell factory" selection were required for the high-Env phenotype. hVLPs showed greater infectivity than standard pseudovirions but largely similar neutralization sensitivity. Importantly, hVLPs also showed superior activation of Env-specific B cells. Hence, high-Env HIV-1 virions, obtained through selection of producer cells, represent an adaptable platform for vaccine design and should aid in the study of native Env.IMPORTANCE The paucity of spikes on HIV is a unique feature that has been associated with evasion of the immune system, while increasing spike density has been a goal of vaccine design. Increasing the density of Env by modifying it in various ways has met with limited success. Here, we focused instead on the producer cell. Cells that stably express HIV spikes were screened on the basis of high binding by bnAbs and low binding by nonneutralizing antibodies. Levels of spikes on cells correlated well with those on progeny virions. Importantly, high-Env virus-like particles (hVLPs) were produced with a manifest array of well-defined spikes, and these were shown to be superior in activating desirable B cells. Our study describes HIV particles that are densely coated with functional spikes, which should facilitate the study of HIV spikes and their development as immunogens.

Nanoparticle conjugation of antigen enhances cytotoxic T-cell responses in pulmonary vaccination.

The ability of vaccines to induce memory cytotoxic T-cell responses in the lung is crucial in stemming and treating pulmonary diseases caused by viruses and bacteria. However, most approaches to subunit vaccines produce primarily humoral and only to a lesser extent cellular immune responses. We developed a nanoparticle (NP)-based carrier that, upon delivery to the lung, specifically targets pulmonary dendritic cells, thus enhancing antigen uptake and transport to the draining lymph node; antigen coupling via a disulfide link promotes highly efficient cross-presentation after uptake, inducing potent protective mucosal and systemic CD8(+) T-cell immunity. Pulmonary immunization with NP-conjugated ovalbumin (NP-ova) with CpG induced a threefold enhancement of splenic antigen-specific CD8(+) T cells displaying increased CD107a expression and IFN-γ production compared with immunization with soluble (i.e., unconjugated) ova with CpG. This enhanced response was accompanied by a potent Th17 cytokine profile in CD4(+) T cells. After 50 d, NP-ova and CpG also led to substantial enhancements in memory CD8(+) T-cell effector functions. Importantly, pulmonary vaccination with NP-ova and CpG induced as much as 10-fold increased frequencies of antigen-specific effector CD8(+) T cells to the lung and completely protected mice from morbidity following influenza-ova infection. Here, we highlight recruitment to the lung of a long-lasting pool of protective effector memory cytotoxic T-cells by our disulfide-linked antigen-conjugated NP formulation. These results suggest the reduction-reversible NP system is a highly promising platform for vaccines specifically targeting intracellular pathogens infecting the lung.

Proof of concept for rational design of hepatitis C virus E2 core nanoparticle vaccines.

Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are responsible for cell entry, with E2 being the major target of neutralizing antibodies (NAbs). Here, we present a comprehensive strategy for B cell-based HCV vaccine development through E2 optimization and nanoparticle display. We redesigned variable region 2 in a truncated form (tVR2) on E2 cores derived from genotypes 1a and 6a, resulting in improved stability and antigenicity. Crystal structures of three optimized E2 cores with human cross-genotype NAbs (AR3s) revealed how the modified tVR2 stabilizes E2 without altering key neutralizing epitopes. We then displayed these E2 cores on 24- and 60-meric nanoparticles and achieved substantial yield and purity, as well as enhanced antigenicity. In mice, these nanoparticles elicited more effective NAb responses than soluble E2 cores. Next-generation sequencing (NGS) defined distinct B cell patterns associated with nanoparticle-induced antibody responses, which target the conserved neutralizing epitopes on E2 and cross-neutralize HCV genotypes.

An MPER antibody neutralizes HIV-1 using germline features shared among donors.

The membrane-proximal external region (MPER) of HIV-1 envelope glycoprotein (Env) can be targeted by neutralizing antibodies of exceptional breadth. MPER antibodies usually have long, hydrophobic CDRH3s, lack activity as inferred germline precursors, are often from the minor IgG3 subclass, and some are polyreactive, such as 4E10. Here we describe an MPER broadly neutralizing antibody from the major IgG1 subclass, PGZL1, which shares germline V/D-region genes with 4E10, has a shorter CDRH3, and is less polyreactive. A recombinant sublineage variant pan-neutralizes a 130-isolate panel at 1.4 µg/ml (IC50). Notably, a germline revertant with mature CDR3s neutralizes 12% of viruses and still binds MPER after DJ reversion. Crystal structures of lipid-bound PGZL1 variants and cryo-EM reconstruction of an Env-PGZL1 complex reveal how these antibodies recognize MPER and viral membrane. Discovery of common genetic and structural elements among MPER antibodies from different patients suggests that such antibodies could be elicited using carefully designed immunogens.

High-Density Array of Well-Ordered HIV-1 Spikes on Synthetic Liposomal Nanoparticles Efficiently Activate B Cells.

A major step toward an HIV-1 vaccine is an immunogen capable of inducing neutralizing antibodies. Envelope glycoprotein (Env) mimetics, such as the NFL and SOSIP designs, generate native-like, well-ordered trimers and elicit tier 2 homologous neutralization (SOSIPs). We reasoned that the display of well-ordered trimers by high-density, particulate array would increase B cell activation compared to soluble trimers. Here, we present the design of liposomal nanoparticles displaying well-ordered Env spike trimers on their surface. Biophysical analysis, cryo- and negative stain electron microscopy, as well as binding analysis with a panel of broadly neutralizing antibodies confirm a high-density, well-ordered trimer particulate array. The Env-trimer-conjugated liposomes were superior to soluble trimers in activating B cells ex vivo and germinal center B cells in vivo. In addition, the trimer-conjugated liposomes elicited modest tier 2 homologous neutralizing antibodies. The trimer-conjugated liposomes represent a promising initial lead toward the development of more effective HIV vaccine immunogens.

A high-throughput nanoimmunoassay chip applied to large-scale vaccine adjuvant screening.

Large-scale experimentation is becoming instrumental in enabling new discoveries in systems biology and personalized medicine. We developed a multiplexed high-throughput nanoimmunoassay chip capable of quantifying four biomarkers in 384 5 nL samples, for a total of 1536 assays. Our platform, compared to conventional methods, reduces volume and reagent cost by ~1000-fold. We applied our platform in the context of systems vaccinology, to assess the synergistic production of inflammatory cytokines from dendritic cells (DCs) stimulated with 10 different adjuvants that target members of the Toll-like receptor (TLR) family. We quantified these adjuvants both alone and in all pairwise combinations, for a total of 435 conditions, revealing numerous synergistic pairs. We evaluated two synergistic interactions, MPLA + Gardiquimod and MPLA + CpG-B, in a mouse model, where we measured the same inflammatory cytokines in bronchoalveolar lavage and in blood serum at 4 different time points using our chip, and observed similar synergistic effects in vivo, demonstrating the potential of our microfluidic platform to predict agonistic immunogenicity. More generally, a high-throughput, matrix-insensitive, low sample volume technology can play an important role in the discovery of novel therapeutics and research areas requiring large-scale biomarker quantitation.

Tunable T cell immunity towards a protein antigen using polymersomes vs. solid-core nanoparticles.

Using poly(propylene sulfide) (PPS) and poly(ethylene glycol) (PEG) as components of a nanocarrier platform, we sought to compare immune responses induced by PPS-bl-PEG polymersomes (PSs; watery-core structures, with antigen incorporated within the PSs) and PEG-stabilized PPS nanoparticles (NPs; solid-core structures, with antigen conjugated upon the NP surface). We have previously shown strong CD8(+) T cell responses to antigen conjugated to NPs via a disulfide link, and here we investigated the extent to which antigen incorporated within oxidatively-sensitive PSs could induce CD4(+) or CD8(+) T cell responses. C57BL/6 mice were subcutaneously immunized with free ovalbumin (OVA) as a model antigen, or equivalent doses of OVA-loaded into PSs, conjugated onto NPs, or given as a mixture of the two. Free CpG was used as an adjuvant. Antigen-loaded PSs induced enhanced frequencies of antigen-specific CD4(+) T cells in the spleen, lymph nodes and lungs as compared to the NP formulation, whereas antigen-conjugated NPs induced stronger CD8(+) T cell responses. Co-administration of both PSs and NPs elicited T cell immunity characteristic of the two nanocarriers at the same time, i.e. both strong CD4(+) and CD8(+) T cell responses. These results have important implications for particulate-based vaccine design and highlight the potential of using different antigen-delivery systems for the induction of both T helper and cytotoxic T lymphocyte immune responses.

Nanoparticle size influences the magnitude and quality of mucosal immune responses after intranasal immunization.

BACKGROUND: The development of nanoparticulate antigen-delivery systems is an important emerging area of vaccinology, being sought to amplify immune responses to recombinant antigens that are poorly immunogenic. Nanoparticle size may play an important role in influencing the activity of such particulate-based adjuvants. METHODS: To explore how the size of nanoparticles that are in the range of many common viruses can modulate the magnitude and quality of mucosal immune responses, the model antigen ovalbumin (OVA) was conjugated to 30 nm or 200 nm polypropylene sulfide nanoparticles (NPs) and administered intranasally to C57BL/6 mice. RESULTS: We show that by increasing the size of the NPs from 30 to 200 nm, OVA was more effectively delivered into both MHC class I and MHC class II-presentation pathways. Intranasal immunization with the 200 nm NPs increased the magnitude of CD4(+) T cell responses in the lungs, as well as systemic and mucosal humoral responses. Most importantly, 200 nm NPs increased the proportion of antigen-specific polyfunctional CD4(+) T cells as compared to 30 nm NPs. CONCLUSIONS: The 200 nm NPs are a very interesting antigen nanocarrier for prophylactic vaccines against mucosal pathogens that require multifunctional CD4(+) T cells for protection. These results contribute to our understanding of how the size of an antigen-conjugated nanoparticle modulates mucosal immune responses to a protein antigen and may be useful to engineer subunit vaccines able to elicit appropriate mucosal immune responses that correlate with protection.

Dendritic cell activation and T cell priming with adjuvant- and antigen-loaded oxidation-sensitive polymersomes.

While current subunit vaccines successfully induce humoral immune responses, a need exists for vaccine strategies to elicit strong cell-mediated immunity to address diseases such as cancer and chronic viral infection. Polymersomes are stable vesicles composed of self-assembling block copolymers with tunable degradation properties allowing delivery of both hydrophilic (within vesicle interior) or hydrophobic (within vesicle membrane) payload molecules. Here we apply oxidation-sensitive nanoscale polymersomes for both antigen and adjuvant delivery to dendritic cell (DC) endosomes. Calcein-loaded polymersomes were observed to release their payload initially in multiple DC endosomal compartments and subsequently within the cytosol. With either the Toll-like receptor agonists gardiquimod or R848 as payloads within the polymersomes, release resulted in DC activation, as indicated by induction of inflammatory cytokine expression and upregulation of DC maturation surface markers: for example, the ability of gardiquimod to induce IL-6 and IL-12 cytokine expression by DCs was enhanced 10-fold when loaded within polymersomes. With the model antigen ovalbumin as a payload, release resulted in CD8(+) T cell cross-priming by promoting protein antigen cross-presentation through MHC I, as indicated by activation of OT-I CD8(+) T cells. Our results demonstrate that oxidation-sensitive polymersomes can function as a vaccine delivery platform for inducing cell-mediated antigen-specific immune responses.

PPS nanoparticles as versatile delivery system to induce systemic and broad mucosal immunity after intranasal administration.

Degradable polymer nanoparticles (NPs, 50 nm) based on polypropylene sulfide (PPS) were conjugated to thiolated antigen and adjuvant proteins by reversible disulfide bonds and evaluated in mucosal vaccination. Ovalbumin was used as a model antigen, and antigen-conjugated NPs were administered intranasally in the mouse. We show penetration of nasal mucosae, transit via M cells, and uptake by antigen-presenting cells in the nasal-associated lymphoid tissue. Ovalbumin-conjugated NPs induced cytotoxic T lymphocytic responses in lung and spleen tissues, as well as humoral response in mucosal airways. Co-conjugation of the TLR5 ligand flagellin further enhanced humoral responses in the airways as well as in the distant vaginal and rectal mucosal compartments and induced cellular immune responses with a Th1 bias, in contrast with free flagellin. The PPS NP platform thus appears interesting as a platform for intranasally-administered mucosal vaccination for inducing broad mucosal immunity.

Study of the genotypic resistant pattern in HIV-infected women and children from rural west Cameroon.

The distribution of antiretroviral (ARV) therapy resistance mutations among HIV-1 strains was evaluated in 39 postpartum women, one pregnant woman, and 12 HIV-positive babies (seven newborns and five children) living in rural west Cameroon. Thirty-five women and all newborns received a single dose of nevirapine (NVP) to prevent mother-to-child transmission of HIV-1; two women were ARV treated and three were ARV naive. Of the 52 viral strains examined, three were subtype B, 45 were classified into eight HIV-1 non-B subtypes, and four remained unclassifiable. Sequence analysis for genotypic drug resistance in the reverse transcriptase (RT) gene showed the presence of mutations associated with nonnucleoside RT inhibitor resistance in 20% of the samples from NVP-treated women and in 57% of those from treated newborns. Mutations associated with nucleoside RT inhibitors (M184V in one case and V118I in four cases) were found in five samples, despite being derived from ARV-naive patients. As expected, a greater frequency of mutations was found in the protease gene region. Of the sequences analyzed, 79% harbored five to seven specific mutations. The secondary mutations showed the typical protease inhibitor resistance-associated pattern for non-subtype B viruses, M36I being the predominant mutation (92.5% in women, 100% in babies). Other mutations frequently detected were K20I, L63P, H69K, and I13V. These findings confirm that resistance mutations can be detected in ARV-naive patients infected with non-B subtypes and emphasize an urgent need for studies assessing the impact of these mutations on the efficacy of subsequent ARV therapy and on the appearance of drug-resistant strains.

Genotypic resistance of archived and circulating viral strains in the blood of treated HIV-infected individuals.

The aim of this study was to evaluate patterns of antiretroviral resistance of HIV-1 in peripheral blood mononuclear cells (PBMCs) and in the plasma of patients whose therapeutic regimen is failing. Plasma and PBMC samples were collected from 95 HIV-infected patients undergoing long-term treatment. Genotyping of the reverse transcriptase (RT) and protease genes of HIV-1 was undertaken using the fluorescent dideoxy-terminator method. Comparison of the amino acid sequence of the RT and protease genes in cell-associated variants of HIV-1 with that of the plasma revealed that 62 of the 95 patients' samples tested exhibited different genotypic resistance patterns (discordant samples [DSs]). In 27% of samples, the patterns of resistance detected were concordant in both compartments. In 51% of DSs, the greatest number of mutations was found in plasma; however, in 37% of DSs, greater numbers of mutations were found in PBMC DNA. The HIV mutation patterns detected in plasma do not necessarily reflect those found in the cell-associated compartment. The observation that the cellular compartment may contain an archive of the resistance variant makes this reservoir an interesting substrate for analysis of the "resistance potential" in a given patient.