A flexible, thermostable nanostructured lipid carrier platform for RNA vaccine delivery.

Current RNA vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are limited by instability of both the RNA and the lipid nanoparticle delivery system, requiring storage at -20°C or -70°C and compromising universally accessible vaccine distribution. This study demonstrates the thermostability and adaptability of a nanostructured lipid carrier (NLC) delivery system for RNA vaccines that has the potential to address these concerns. Liquid NLC alone is stable at refrigerated temperatures for ≥1 year, enabling stockpiling and rapid deployment by point-of-care mixing with any vaccine RNA. Alternatively, NLC complexed with RNA may be readily lyophilized and stored at room temperature for ≥8 months or refrigerated temperature for ≥21 months while still retaining the ability to express protein in vivo. The thermostability of this NLC/RNA vaccine delivery platform could significantly improve distribution of current and future pandemic response vaccines, particularly in low-resource settings.

Live-attenuated RNA hybrid vaccine technology provides single-dose protection against Chikungunya virus.

We present a live-attenuated RNA hybrid vaccine technology that uses an RNA vaccine delivery vehicle to deliver in vitro-transcribed, full-length, live-attenuated viral genomes to the site of vaccination. This technology allows ready manufacturing in a cell-free environment, regardless of viral attenuation level, and it promises to avoid many safety and manufacturing challenges of traditional live-attenuated vaccines. We demonstrate this technology through development and testing of a live-attenuated RNA hybrid vaccine against Chikungunya virus (CHIKV), comprised of an in vitro-transcribed, highly attenuated CHIKV genome delivered by a highly stable nanostructured lipid carrier (NLC) formulation as an intramuscular injection. We demonstrate that single-dose immunization of immunocompetent C57BL/6 mice results in induction of high CHIKV-neutralizing antibody titers and protection against mortality and footpad swelling after lethal CHIKV challenge.

Integrated pipeline for the accelerated discovery of antiviral antibody therapeutics.

The emergence and re-emergence of highly virulent viral pathogens with the potential to cause a pandemic creates an urgent need for the accelerated discovery of antiviral therapeutics. Antiviral human monoclonal antibodies (mAbs) are promising candidates for the prevention and treatment of severe viral diseases, but their long development timeframes limit their rapid deployment and use. Here, we report the development of an integrated sequence of technologies, including single-cell mRNA-sequence analysis, bioinformatics, synthetic biology and high-throughput functional analysis, that enables the rapid discovery of highly potent antiviral human mAbs, the activity of which we validated in vivo. In a 78-d study modelling the deployment of a rapid response to an outbreak, we isolated more than 100 human mAbs that are specific to Zika virus, assessed their function, identified that 29 of these mAbs have broadly neutralizing activity, and verified the therapeutic potency of the lead candidates in mice and non-human primate models of infection through the delivery of an antibody-encoding mRNA formulation and of the respective IgG antibody. The pipeline provides a roadmap for rapid antibody-discovery programmes against viral pathogens of global concern.

Intramuscular Delivery of Replicon RNA Encoding ZIKV-117 Human Monoclonal Antibody Protects against Zika Virus Infection.

Monoclonal antibody (mAb) therapeutics are an effective modality for the treatment of infectious, autoimmune, and cancer-related diseases. However, the discovery, development, and manufacturing processes are complex, resource-consuming activities that preclude the rapid deployment of mAbs in outbreaks of emerging infectious diseases. Given recent advances in nucleic acid delivery technology, it is now possible to deliver exogenous mRNA encoding mAbs for in situ expression following intravenous (i.v.) infusion of lipid nanoparticle-encapsulated mRNA. However, the requirement for i.v. administration limits the application to settings where infusion is an option, increasing the cost of treatment. As an alternative strategy, and to enable intramuscular (IM) administration of mRNA-encoded mAbs, we describe a nanostructured lipid carrier for delivery of an alphavirus replicon encoding a previously described highly neutralizing human mAb, ZIKV-117. Using a lethal Zika virus challenge model in mice, our studies show robust protection following alphavirus-driven expression of ZIKV-117 mRNA when given by IM administration as pre-exposure prophylaxis or post-exposure therapy.

Vaccine adjuvant activity of emulsified oils from species of the Pinaceae family.

BACKGROUND: Next to aluminum salts, squalene nanoemulsions comprise the most widely employed class of adjuvants in approved vaccines. Despite their importance, the mechanisms of action of squalene nanoemulsions are not completely understood, nor are the structure/function requirements of the oil composition. PURPOSE: In this study, we build on previous work that compared the adjuvant properties of nanoemulsions made with different classes of oil structures to squalene nanoemulsion. Here, we introduce nanoemulsions made with polyprenols derived from species of the Pinaceae family as novel vaccine adjuvant compositions. In contrast with long-chain triglycerides that do not efficiently enhance an immune response, both polyprenols and squalene are comprised of multimeric isoprene units, which may represent an important structural property of oils in nanoemulsions with adjuvant properties. STUDY DESIGN: Oils derived from species of the Pinaceae family were formulated in nanoemulsions, with or without a synthetic Toll-like receptor 4 (TLR4) ligand, and characterized regarding physicochemical and biological activity properties in comparison to squalene nanoemulsions. METHODS: Oils were extracted from species of the Pinaceae family and used to prepare oil-in-water nanoemulsions by microfluidization. Emulsion droplet diameter stability was characterized by dynamic light scattering. Nanoemulsions were evaluated for in vitro biological activity using human whole blood, and in vivo biological activity in mouse, pig, and ferret models when combined with pandemic influenza vaccine antigens. RESULTS: Nanoemulsions comprised of Pinaceae-derived polyprenol oils demonstrated long-term physical stability, stimulated cytokine production from human cells in vitro, and promoted antigen-specific immune responses in various animal models, particularly when formulated with the TLR4 ligand glucopyranosyl lipid adjuvant (GLA). CONCLUSION: Pinaceae-derived nanoemulsions are compatible with inclusion of a synthetic TLR4 ligand and promote antigen-specific immune responses to pandemic influenza antigens in mouse, pig, and ferret models.

Glucopyranosyl lipid adjuvant enhances immune response to Ebola virus-like particle vaccine in mice.

The identification of adjuvants that promote lasting antigen-specific immunity and augment vaccine efficacy are integral to the development of new protein-based vaccines. The Ebola virus-like particle (VLP) vaccine expressing Ebola virus glycoprotein (GP) and matrix protein (VP40) was used in this study to evaluate the ability of TLR4 agonist glucopyranosyl lipid adjuvant (GLA) formulated in a stable emulsion (SE) to enhance immunogenicity and promote durable protection against mouse-adapted Ebola virus (ma-EBOV). Antibody responses and Ebola-specific T cell responses were evaluated post vaccination. Survival analysis after lethal ma-EBOV challenge was performed 4 weeks and 22 weeks following final vaccination. GLA-SE enhanced EBOV-specific immunity and resulted in long-term protection against challenge with ma-EBOV infection in a mouse model. Specifically, GLA-SE elicited Th1-skewed antibodies and promoted the generation of EBOV GP-specific polyfunctional T cells. These results provide further support for the utility of TLR4 activating GLA-SE-adjuvanted vaccines.

Memory CD4+ T cells enhance B-cell responses to drifting influenza immunization.

Influenza A annually infects 5-10% of the world's human population resulting in one million deaths. Influenza causes annual epidemics and reinfects previously exposed individuals because of antigenic drift in the glycoprotein hemagglutinin. Due to antigenic drift, the immune system is simultaneously exposed to novel and conserved parts of the influenza virus via vaccination and/or infection throughout life. Preexisting immunity has long been known to augment subsequent hemagglutination inhibitory antibody (hAb) responses. However, the preexisting immunological contributors that influence hAb responses are not understood. Therefore, we adapted and developed sequential infection and immunization mouse models using drifted influenza strains to show that MHC Class II haplotype and T-cell reactivity influences subsequent hAb responses. We found that CB6F1 mice infected with A/CA followed by immunization with A/PR8 have increased hAb responses to A/PR8 compared to C57BL/6 mice. Increased hAb responses in CB6F1 mice were CD4+  T-cell and B-cell dependent and corresponded to increased germinal center A/PR8-specific B and T-follicular helper cells. These results suggest conserved MHC Class II restricted epitopes within HA are essential for B cells to respond to drifting influenza and could be leveraged to boost hAb responses.

Heterologous Immunization With Defined RNA and Subunit Vaccines Enhances T Cell Responses That Protect Against Leishmania donovani.

The rapid generation of strong T cell responses is highly desirable and viral vectors can have potent CD8+ T cell-inducing activity. Immunity to leishmaniasis requires selective T cell responses, with immunization schemes that raise either CD4 or CD8 T cell responses being protective in small animal models. We have defined the leishmaniasis vaccine candidate recombinant fusion antigens, LEISH-F2 and LEISH-F3+, that when formulated in a stable emulsion with a Toll-like receptor (TLR) 4 agonist, induce protective CD4+ T cell responses in animal models as well as providing therapeutic efficacy in canine leishmaniasis and in clinical trials in leishmaniasis patients. We used the genetic sequences of these validated vaccine antigens to design RNA vaccine constructs. Immunization of mice with the RNA replicons induced potent, local innate responses that were surprisingly independent of TLR7 and activated antigen-presenting cells (APC) to prime for extremely potent antigen-specific T helper 1 type responses upon heterologous boosting with either of the subunit vaccines (recombinant antigen with second generation glucopyranosyl lipid A in stable oil-in-water emulsion; SLA-SE). Inclusion of RNA in the immunization schedule also generated MHCI-restricted T cell responses. Immunization with LEISH-F2-expressing RNA vaccine followed later by subunit vaccine afforded protection against challenge with Leishmania donovani. Together, these data indicate the utility of heterologous prime-boost immunization schemes for the induction of potent antigen-specific CD4 and CD8 T cell responses for protection against intracellular pathogens.

A combination of TLR-4 agonist and saponin adjuvants increases antibody diversity and protective efficacy of a recombinant West Nile Virus antigen.

Members of the Flaviviridae family are the leading causes of mosquito-borne viral disease worldwide. While dengue virus is the most prevalent, the recent Zika virus outbreak in the Americas triggered a WHO public health emergency, and yellow fever and West Nile viruses (WNV) continue to cause regional epidemics. Given the sporadic nature of flaviviral epidemics both temporally and geographically, there is an urgent need for vaccines that can rapidly provide effective immunity. Protection from flaviviral infection is correlated with antibodies to the viral envelope (E) protein, which encodes receptor binding and fusion functions. TLR agonist adjuvants represent a promising tool to enhance the protective capacity of flavivirus vaccines through dose and dosage reduction and broadening of antiviral antibody responses. This study investigates the ability to improve the immunogenicity and protective capacity of a promising clinical-stage WNV recombinant E-protein vaccine (WN-80E) using a novel combination adjuvant, which contains a potent TLR-4 agonist and the saponin QS21 in a liposomal formulation (SLA-LSQ). Here, we show that, in combination with WN-80E, optimized SLA-LSQ is capable of inducing long-lasting immune responses in preclinical models that provide sterilizing protection from WNV challenge, reducing viral titers following WNV challenge to undetectable levels in Syrian hamsters. We have investigated potential mechanisms of action by examining the antibody repertoire generated post-immunization. SLA-LSQ induced a more diverse antibody response to WNV recombinant E-protein antigen than less protective adjuvants. Collectively, these studies identify an adjuvant formulation that enhances the protective capacity of recombinant flavivirus vaccines.

The adjuvant GLA-AF enhances human intradermal vaccine responses.

Adjuvants are key to shaping the immune response to vaccination, but to date, no adjuvant suitable for human use has been developed for intradermal vaccines. These vaccines could be self-administered and sent through the mail as they do not require long needles or technical expertise in immunization. In the event of a pandemic outbreak, this approach could alleviate the congregation of patients in health centers and thus reduce the potential of these centers to enhance the spread of lethal infection. A reliable and potent vaccine system for self-administration would provide an effective countermeasure for delivery through existing product distribution infrastructure. We report results from preclinical and clinical trials that demonstrate the feasibility of an adjuvanted, intradermal vaccine that induced single shot protection in ferrets and seroprotection in humans against one of the more lethal strains of pandemic flu, Indonesia H5N1. In the human trial, the vaccine was safe and clinical responses were above approvable endpoints for a protective flu vaccine. Inclusion of a modern TLR4 (Toll-like receptor 4) agonist-based adjuvant was critical to the development of the response in the intradermal groups. In humans, this is the first report of a safe and effective intradermal adjuvant, GLA-AF (aqueous formulation of glucopyranosyl lipid adjuvant), and provides a future path for developing a vaccine-device combination for distribution by mail and self-administration in case of a pandemic.

A Nanostructured Lipid Carrier for Delivery of a Replicating Viral RNA Provides Single, Low-Dose Protection against Zika.

Since the first demonstration of in vivo gene expression from an injected RNA molecule almost two decades ago,1 the field of RNA-based therapeutics is now taking significant strides, with many cancer and infectious disease targets entering clinical trials.2 Critical to this success has been advances in the knowledge and application of delivery formulations. Currently, various lipid nanoparticle (LNP) platforms are at the forefront,3 but the encapsulation approach underpinning LNP formulations offsets the synthetic and rapid-response nature of RNA vaccines.4 Second, limited stability of LNP formulated RNA precludes stockpiling for pandemic readiness.5 Here, we show the development of a two-vialed approach wherein the delivery formulation, a highly stable nanostructured lipid carrier (NLC), can be manufactured and stockpiled separate from the target RNA, which is admixed prior to administration. Furthermore, specific physicochemical modifications to the NLC modulate immune responses, either enhancing or diminishing neutralizing antibody responses. We have combined this approach with a replicating viral RNA (rvRNA) encoding Zika virus (ZIKV) antigens and demonstrated a single dose as low as 10 ng can completely protect mice against a lethal ZIKV challenge, representing what might be the most potent approach to date of any Zika vaccine.

Improved Immune Responses in Young and Aged Mice with Adjuvanted Vaccines against H1N1 Influenza Infection.

Elderly people are at high risk for influenza-related morbidity and mortality due to progressive immunosenescence. While toll-like receptor (TLR) agonist containing adjuvants, and other adjuvants, have been shown to enhance influenza vaccine-induced protective responses, the mechanisms underlying how these adjuvanted vaccines could benefit the elderly remain elusive. Here, we show that a split H1N1 influenza vaccine (sH1N1) combined with a TLR4 agonist, glucopyranosyl lipid adjuvant formulated in a stable oil-in-water emulsion (GLA-SE), boosts IgG2c:IgG1 ratios, enhances hemagglutination inhibition (HAI) titers, and increases protection in aged mice. We find that all adjuvanted sH1N1 vaccines tested were able to protect both young and aged mice from lethal A/H1N1/California/4/2009 virus challenge after two immunizations compared to vaccine alone. We show that GLA-SE combined with sH1N1, however, also provides enhanced protection from morbidity in aged mice given one immunization (based on change in weight percentage). While the GLA-SE-adjuvanted sH1N1 vaccine promotes the generation of cytokine-producing T helper 1 cells, germinal center B cells, and long-lived bone marrow plasma cells in young mice, these responses were muted in aged mice. Differential in vitro responses, dependent on age, were also observed from mouse-derived bone marrow-derived dendritic cells and lung homogenates following stimulation with adjuvants, including GLA-SE. Besides enhanced HAI titers, additional protective factors elicited with sH1N1 + GLA-SE in young mice were observed, including (a) rapid reduction of viral titers in the lung, (b) prevention of excessive lung inflammation, and (c) homeostatic maintenance of alveolar macrophages (AMs) following H1N1 infection. Collectively, our results provide insight into mechanisms of adjuvant-mediated immune protection in the young and elderly.

A Formulated TLR7/8 Agonist is a Flexible, Highly Potent and Effective Adjuvant for Pandemic Influenza Vaccines.

Since 1997, highly pathogenic avian influenza viruses of the H5N1 subtype have been transmitted from avian hosts to humans. The severity of H5N1 infection in humans, as well as the sporadic nature of H5N1 outbreaks, both geographically and temporally, make generation of an effective vaccine a global public health priority. An effective H5N1 vaccine must ultimately provide protection against viruses from diverse clades. Toll-like receptor (TLR) agonist adjuvant formulations have a demonstrated ability to broaden H5N1 vaccine responses in pre-clinical models. However, many of these agonist molecules have proven difficult to develop clinically. Here, we describe comprehensive adjuvant formulation development of the imidazoquinoline TLR-7/8 agonist 3M-052, in combination with H5N1 hemagglutinin (HA) based antigens. We find that 3M-052 in multiple formulations protects both mice and ferrets from lethal H5N1 homologous virus challenge. Furthermore, we conclusively demonstrate the ability of 3M-052 adjuvant formulations to broaden responses to H5N1 HA based antigens, and show that this broadening is functional using a heterologous lethal virus challenge in ferrets. Given the extensive clinical use of imidazoquinoline TLR agonists for other indications, these studies identify multiple adjuvant formulations which may be rapidly advanced into clinical trials in an H5N1 vaccine.

Adsorption of a synthetic TLR7/8 ligand to aluminum oxyhydroxide for enhanced vaccine adjuvant activity: A formulation approach.

For nearly a century, aluminum salts have been the most widely used vaccine adjuvant formulation, and have thus established a history of safety and efficacy. Nevertheless, for extremely challenging disease targets such as tuberculosis or HIV, the adjuvant activity of aluminum salts may not be potent enough to achieve protective efficacy. Adsorption of TLR ligands to aluminum salts facilitates enhanced adjuvant activity, such as in the human papilloma virus vaccine Cervarix®. However, some TLR ligands such as TLR7/8 agonist imidazoquinolines do not efficiently adsorb to aluminum salts. The present report describes a formulation approach to solving this challenge by developing a lipid-based nanosuspension of a synthetic TLR7/8 ligand (3M-052) that facilitates adsorption to aluminum oxyhydroxide via the structural properties of the helper lipid employed. In immunized mice, the aluminum oxyhydroxide-adsorbed formulation of 3M-052 enhanced antibody and TH1-type cellular immune responses to vaccine antigens for tuberculosis and HIV.

Optimizing Immunization Strategies for the Induction of Antigen-Specific CD4 and CD8 T Cell Responses for Protection against Intracellular Parasites.

Immunization strategies that generate either CD4 or CD8 T cell responses are relatively well described, but less is known with regard to optimizing regimens to induce both CD4 and CD8 memory T cells. Considering the importance of both CD4 and CD8 T cells in the control of intracellular pathogens such as Leishmania donovani, we wanted to identify vaccines that could raise both CD4 and CD8 T cell responses and determine how to configure immunization strategies to generate the best combined protective T cell response. We examined responses generated against the Leishmania vaccine antigen F3 following its administration in either recombinant form with the Toll-like receptor 4 (TLR4) agonist-containing adjuvant formulation GLA-SE (F3+GLA-SE) or as a gene product delivered in an adenoviral vector (Ad5-F3). Homologous immunization strategies using only F3+GLA-SE or Ad5-F3 preferentially generated either CD4 or CD8 T cells, respectively. In contrast, heterologous strategies generated both antigen-specific CD4 and CD8 T cells. Administration of F3+GLA-SE before Ad5-F3 generated the greatest combined CD4 and CD8 responses. Cytotoxic CD8 T cell responses were highest when Th1 cells were generated prior to their induction by Ad5-F3. Finally, a single immunization with a combination of F3+GLA-SE mixed with Ad5-F3 was found to be sufficient to provide protection against experimental L. donovani infection. Taken together, our data delineate immunization regimens that induce antigen-specific CD4 and CD8 T cell memory responses, and identify a single immunization strategy that could be used to rapidly provide protection against intracellular pathogens in regions where access to health care is limited or sporadic.

Selection of therapeutic H5N1 monoclonal antibodies following IgVH repertoire analysis in mice.

The rapid rate of influenza virus mutation drives the emergence of new strains that inflict serious seasonal epidemics and less frequent, but more deadly, pandemics. While vaccination provides the best protection against influenza, its utility is often diminished by the unpredictability of new pathogenic strains. Consequently, efforts are underway to identify new antiviral drugs and monoclonal antibodies that can be used to treat recently infected individuals and prevent disease in vulnerable populations. Next Generation Sequencing (NGS) and the analysis of antibody gene repertoires is a valuable tool for Ab discovery. Here, we describe a technology platform for isolating therapeutic monoclonal antibodies (MAbs) by analyzing the IgVH repertoires of mice immunized with recombinant H5N1 hemagglutinin (rH5). As an initial proof of concept, 35 IgVH genes were selected using a CDRH3 search algorithm and co-expressed in a murine IgG2a expression vector with a panel of germline murine kappa genes. Culture supernatants were then screened for antigen binding. Seventeen of the 35 IgVH MAbs (49%) bound rH5VN1203 in preliminary screens and 8 of 9 purified MAbs inhibited 3 heterosubtypic strains of H5N1 virus when assayed by HI. Two of these MAbs demonstrated prophylactic and therapeutic activity in virus-challenged mice. This is the first example in which an NGS discovery platform has been used to isolate anti-influenza MAbs with relevant therapeutic activity.

A Novel Synthetic TLR-4 Agonist Adjuvant Increases the Protective Response to a Clinical-Stage West Nile Virus Vaccine Antigen in Multiple Formulations.

West Nile virus (WNV) is a mosquito-transmitted member of the Flaviviridae family that has emerged in recent years to become a serious public health threat. Given the sporadic nature of WNV epidemics both temporally and geographically, there is an urgent need for a vaccine that can rapidly provide effective immunity. Protection from WNV infection is correlated with antibodies to the viral envelope (E) protein, which encodes receptor binding and fusion functions. Despite many promising E-protein vaccine candidates, there are currently none licensed for use in humans. This study investigates the ability to improve the immunogenicity and protective capacity of a promising clinical-stage WNV recombinant E-protein vaccine (WN-80E) by combining it with a novel synthetic TLR-4 agonist adjuvant. Using the murine model of WNV disease, we find that inclusion of a TLR-4 agonist in either a stable oil-in-water emulsion (SE) or aluminum hydroxide (Alum) formulation provides both dose and dosage sparing functions, whereby protection can be induced after a single immunization containing only 100 ng of WN-80E. Additionally, we find that inclusion of adjuvant with a single immunization reduced viral titers in sera to levels undetectable by viral plaque assay. The enhanced protection provided by adjuvanted immunization correlated with induction of a Th1 T-cell response and the resultant shaping of the IgG response. These findings suggest that inclusion of a next generation adjuvant may greatly enhance the protective capacity of WNV recombinant subunit vaccines, and establish a baseline for future development.

Technology transfer of oil-in-water emulsion adjuvant manufacturing for pandemic influenza vaccine production in Romania: Preclinical evaluation of split virion inactivated H5N1 vaccine with adjuvant.

Millions of seasonal and pandemic influenza vaccine doses containing oil-in-water emulsion adjuvant have been administered in order to enhance and broaden immune responses and to facilitate antigen sparing. Despite the enactment of a Global Action Plan for Influenza Vaccines and a multi-fold increase in production capabilities over the past 10 years, worldwide capacity for pandemic influenza vaccine production is still limited. In developing countries, where routine influenza vaccination is not fully established, additional measures are needed to ensure adequate supply of pandemic influenza vaccines without dependence on the shipment of aid from other, potentially impacted first-world countries. Adaptation of influenza vaccine and adjuvant technologies by developing country influenza vaccine manufacturers may enable antigen sparing and corresponding increases in global influenza vaccine coverage capacity. Following on previously described work involving the technology transfer of oil-in-water emulsion adjuvant manufacturing to a Romanian vaccine manufacturing institute, we herein describe the preclinical evaluation of inactivated split virion H5N1 influenza vaccine with emulsion adjuvant, including immunogenicity, protection from virus challenge, antigen sparing capacity, and safety. In parallel with the evaluation of the bioactivity of the tech-transferred adjuvant, we also describe the impact of concurrent antigen manufacturing optimization activities. Depending on the vaccine antigen source and manufacturing process, inclusion of adjuvant was shown to enhance and broaden functional antibody titers in mouse and rabbit models, promote protection from homologous virus challenge in ferrets, and facilitate antigen sparing. Besides scientific findings, the operational lessons learned are delineated in order to facilitate adaptation of adjuvant technologies by other developing country institutes to enhance global pandemic influenza preparedness.

Design of a novel integration-deficient lentivector technology that incorporates genetic and posttranslational elements to target human dendritic cells.

As sentinels of the immune system, dendritic cells (DCs) play an essential role in regulating cellular immune responses. One of the main challenges of developing DC-targeted therapies includes the delivery of antigen to DCs in order to promote the activation of antigen-specific effector CD8 T cells. With the goal of creating antigen-directed immunotherapeutics that can be safely administered directly to patients, Immune Design has developed a platform of novel integration-deficient lentiviral vectors that target and deliver antigen-encoding nucleic acids to human DCs. This platform, termed ID-VP02, utilizes a novel genetic variant of a Sindbis virus envelope glycoprotein with posttranslational carbohydrate modifications in combination with Vpx, a SIVmac viral accessory protein, to achieve efficient targeting and transduction of human DCs. In addition, ID-VP02 incorporates safety features in its design that include two redundant mechanisms to render ID-VP02 integration-deficient. Here, we describe the characteristics that allow ID-VP02 to specifically transduce human DCs, and the advances that ID-VP02 brings to conventional third-generation lentiviral vector design as well as demonstrate upstream production yields that will enable manufacturing feasibility studies to be conducted.

Development of a high density hemagglutinin protein microarray to determine the breadth of influenza antibody responses.

We have developed an influenza hemagglutinin protein microarray to assess humoral recognition of diverse influenza strains induced by vaccination and infection. Each array consists of controls and 127 hemagglutinin antigens from 60 viruses, spotted in replicates to generate a single array of 1296 spots. Six arrays are configured on a single slide, which in the following analysis was probed simultaneously with 2 isotype-specific fluorescent secondary antibodies yielding over 15,000 data points per slide. Here we report the use of this system to evaluate mouse, ferret, and human sera. The array allows simultaneous examination of the magnitude of antibody responses, the isotype of such antibodies, and the breadth of influenza strain recognition. We are advancing this technology as a platform for rapid, simple, high-throughput assessment of homologous and heterologous antibody responses to influenza disease and vaccination.