Evaluation of a stabilized RSV pre-fusion F mRNA vaccine: Preclinical studies and Phase 1 clinical testing in healthy adults.

Human respiratory syncytial virus (RSV) causes a substantial proportion of respiratory tract infections worldwide. Although RSV reinfections occur throughout life, older adults, particularly those with underlying comorbidities, are at risk for severe complications from RSV. There is no RSV vaccine available to date, and treatment of RSV in adults is largely supportive. A correlate of protection for RSV has not yet been established, but antibodies targeting the pre-fusion conformation of the RSV F glycoprotein play an important role in RSV neutralization. We previously reported a Phase 1 study of an mRNA-based vaccine (V171) expressing a pre-fusion-stabilized RSV F protein (mDS-Cav1) in healthy adults. Here, we evaluated an mRNA-based vaccine (V172) expressing a further stabilized RSV pre-fusion F protein (mVRC1). mVRC1 is a single chain version of RSV F with interprotomer disulfides in addition to the stabilizing mutations present in the mDS-Cav1 antigen. The immunogenicity of the two mRNA-based vaccines encoding mVRC1 (V172) or a sequence-optimized version of mDS-Cav1 to improve transcriptional fidelity (V171.2) were compared in RSV-naïve and RSV-experienced African green monkeys (AGMs). V172 induced higher neutralizing antibody titers than V171.2 and demonstrated protection in the AGM challenge model. We conducted a Phase 1, randomized, placebo-controlled, clinical trial of 25 µg, 100 µg, 200 µg, or 300 µg of V172 in healthy older adults (60-79 years old; N = 112) and 100 µg, 200 µg, or 300 µg of V172 in healthy younger adults (18-49 years old; N = 48). The primary clinical objectives were to evaluate the safety and tolerability of V172, and the secondary objective was to evaluate RSV serum neutralization titers. The most commonly reported solicited adverse events were injection-site pain, injection-site swelling, headache, and tiredness. V172 was generally well tolerated in older and younger adults and increased serum neutralizing antibody titers, pre-fusion F-specific competing antibody titers, and RSV F-specific T-cell responses.

Split-Dose Administration Enhances Immune Responses Elicited by a mRNA/Lipid Nanoparticle Vaccine Expressing Respiratory Syncytial Virus F Protein.

mRNA vaccines have recently received significant attention due to their role in combating the SARS-CoV-2 pandemic. As a platform, mRNA vaccines have been shown to elicit strong humoral and cellular immune responses with acceptable safety profiles for prophylactic use. Despite their potential, industrial challenges have limited realization of the vaccine platform on a global scale. Critical among these challenges are supply chain considerations, including mRNA production, cost of goods, and vaccine frozen-chain distribution. Here, we assess the delivery of lipid nanoparticle-encapsulated mRNA (mRNA/LNP) vaccines using a split-dose immunization regimen as an approach to develop mRNA dose-sparing vaccine regimens with potential to mitigate mRNA supply chain challenges. Our data demonstrate that immunization by a mRNA/LNP vaccine encoding respiratory syncytial virus pre-F (RSV pre-F) over a 9 day period elicits comparable or superior magnitude of antibodies when compared to traditional bolus immunization of the vaccine. The split-dose immunization regimens evaluated in our studies were designed to mimic reported drug or antigen release profiles from microneedle patches, highlighting the potential benefit of pairing mRNA vaccines with patch-based delivery technologies to enable sustained release and solid-state stabilization. Overall, our findings provide a proof of concept to support further investigations into the development of sustained delivery approaches for mRNA/LNP vaccines.

Preclinical immunogenicity and efficacy of a candidate COVID-19 vaccine based on a vesicular stomatitis virus-SARS-CoV-2 chimera.

BACKGROUND: To investigate a vaccine technology with potential to protect against coronavirus disease 2019 (COVID-19) and reduce transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with a single vaccine dose, we developed a SARS-CoV-2 candidate vaccine using the live vesicular stomatitis virus (VSV) chimeric virus approach previously used to develop a licensed Ebola virus vaccine. METHODS: We generated a replication-competent chimeric VSV-SARS-CoV-2 vaccine candidate by replacing the VSV glycoprotein (G) gene with coding sequence for the SARS-CoV-2 Spike glycoprotein (S). Immunogenicity of the lead vaccine candidate (VSV∆G-SARS-CoV-2) was evaluated in cotton rats and golden Syrian hamsters, and protection from SARS-CoV-2 infection also was assessed in hamsters. FINDINGS: VSV∆G-SARS-CoV-2 delivered with a single intramuscular (IM) injection was immunogenic in cotton rats and hamsters and protected hamsters from weight loss following SARS-CoV-2 challenge. When mucosal vaccination was evaluated, cotton rats did not respond to the vaccine, whereas mucosal administration of VSV∆G-SARS-CoV-2 was found to be more immunogenic than IM injection in hamsters and induced immunity that significantly reduced SARS-CoV-2 challenge virus loads in both lung and nasal tissues. INTERPRETATION: VSV∆G-SARS-CoV-2 delivered by IM injection or mucosal administration was immunogenic in golden Syrian hamsters, and both vaccination methods effectively protected the lung from SARS-CoV-2 infection. Hamsters vaccinated by mucosal application of VSV∆G-SARS-CoV-2 also developed immunity that controlled SARS-CoV-2 replication in nasal tissue. FUNDING: The study was funded by Merck Sharp & Dohme, Corp., a subsidiary of Merck & Co., Inc., Rahway, NJ, USA, and The International AIDS Vaccine Initiative, Inc. (IAVI), New York, USA. Parts of this research was supported by the Biomedical Advanced Research and Development Authority (BARDA) and the Defense Threat Reduction Agency (DTRA) of the US Department of Defense.

Profiling of hMPV F-specific antibodies isolated from human memory B cells.

Human metapneumovirus (hMPV) belongs to the Pneumoviridae family and is closely related to respiratory syncytial virus (RSV). The surface fusion (F) glycoprotein mediates viral fusion and is the primary target of neutralizing antibodies against hMPV. Here we report 113 hMPV-F specific monoclonal antibodies (mAbs) isolated from memory B cells of human donors. We characterize the antibodies' germline usage, epitopes, neutralization potencies, and binding specificities. We find that unlike RSV-F specific mAbs, antibody responses to hMPV F are less dominant against the apex of the antigen, and the majority of the potent neutralizing mAbs recognize epitopes on the side of hMPV F. Furthermore, neutralizing epitopes that differ from previously defined antigenic sites on RSV F are identified, and multiple binding modes of site V and II mAbs are discovered. Interestingly, mAbs that bind preferentially to the unprocessed prefusion F show poor neutralization potency. These results elucidate the immune recognition of hMPV infection and provide novel insights for future hMPV antibody and vaccine development.

Upper and lower respiratory tract correlates of protection against respiratory syncytial virus following vaccination of nonhuman primates.

Respiratory syncytial virus (RSV) infection is a major cause of respiratory illness in infants and the elderly. Although several vaccines have been developed, none have succeeded in part due to our incomplete understanding of the correlates of immune protection. While both T cells and antibodies play a role, emerging data suggest that antibody-mediated mechanisms alone may be sufficient to provide protection. Therefore, to map the humoral correlates of immunity against RSV, antibody responses across six different vaccines were profiled in a highly controlled nonhuman primate-challenge model. Viral loads were monitored in both the upper and lower respiratory tracts, and machine learning was used to determine the vaccine platform-agnostic antibody features associated with protection. Upper respiratory control was associated with virus-specific IgA levels, neutralization, and complement activity, whereas lower respiratory control was associated with Fc-mediated effector mechanisms. These findings provide critical compartment-specific insights toward the rational development of future vaccines.

Modified mRNA/lipid nanoparticle-based vaccines expressing respiratory syncytial virus F protein variants are immunogenic and protective in rodent models of RSV infection.

The RSV Fusion (F) protein is a target for neutralizing antibody responses and is a focus for vaccine discovery; however, the process of RSV entry requires F to adopt a metastable prefusion form and transition to a more stable postfusion form, which displays less potent neutralizing epitopes. mRNA vaccines encode antigens that are translated by host cells following vaccination, which may allow conformational transitions similar to those observed during natural infection to occur. Here we evaluate a panel of chemically modified mRNA vaccines expressing different forms of the RSV F protein, including secreted, membrane associated, prefusion-stabilized, and non-stabilized structures, for conformation, immunogenicity, protection, and safety in rodent models. Vaccination with mRNA encoding native RSV F elicited antibody responses to both prefusion- and postfusion-specific epitopes, suggesting that this antigen may adopt both conformations in vivo. Incorporating prefusion stabilizing mutations further shifts the immune response toward prefusion-specific epitopes, but does not impact neutralizing antibody titer. mRNA vaccine candidates expressing either prefusion stabilized or native forms of RSV F protein elicit robust neutralizing antibody responses in both mice and cotton rats, similar to levels observed with a comparable dose of adjuvanted prefusion stabilized RSV F protein. In contrast to the protein subunit vaccine, mRNA-based vaccines elicited robust CD4+ and CD8+ T-cell responses in mice, highlighting a potential advantage of the technology for vaccines requiring a cellular immune response for efficacy.

Characterization of potent RSV neutralizing antibodies isolated from human memory B cells and identification of diverse RSV/hMPV cross-neutralizing epitopes.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children and older adults. Currently, no licensed vaccine is available, and therapeutic options are limited. The primary target of neutralizing antibodies to RSV is the surface fusion (F) glycoprotein. Understanding the recognition of antibodies with high neutralization potencies to RSV F antigen will provide critical insights in developing efficacious RSV antibodies and vaccines. In this study, we isolated and characterized a panel of monoclonal antibodies (mAbs) with high binding affinity to RSV prefusion F trimer and neutralization potency to RSV viruses. The mAbs were mapped to previously defined antigenic sites, and some that mapped to the same antigenic sites showed remarkable diversity in specificity, binding, and neutralization potencies. We found that the isolated site III mAbs shared highly conserved germline V-gene usage, but had different cross-reactivities to human metapneumovirus (hMPV), possibly due to the distinct modes/angles of interaction with RSV and hMPV F proteins. Furthermore, we identified a subset of potent RSV/hMPV cross-neutralizing mAbs that target antigenic site IV and the recently defined antigenic site V, while the majority of the mAbs targeting these two sites only neutralize RSV. Additionally, the isolated mAbs targeting site Ø were mono-specific for RSV and showed a wide range of neutralizing potencies on different RSV subtypes. Our data exemplify the diversity of anti-RSV mAbs and provide new insights into the immune recognition of respiratory viruses in the Pneumoviridae family.

Immune response and reactogenicity of an unadjuvanted intradermally delivered human papillomavirus vaccine using a first generation Nanopatch™ in rhesus macaques: An exploratory, pre-clinical feasibility assessment.

The human papillomavirus (HPV) 9-valent, recombinant vaccine (Gardasil™9) helps protect young adults (males and females) against anogenital cancers and genital warts caused by certain HPV genotypes (ref. Gardasil™9 insert). This vaccine is administered intramuscularly (IM). The aim of this study was to determine preclinically whether intradermal (ID) vaccination with an unadjuvanted 9-valent recombinant HPV vaccine using a first-generation ID delivery device, the Nanopatch™, could enhance vaccine immunogenicity compared with the traditional ID route (Mantoux technique). IM injection of HPV VLPs formulated with Merck & Co., Inc., Kenilworth, NJ, USA Alum Adjuvant (MAA) were included in the rhesus study for comparison. The Nanopatch™ prototype contains a high-density array comprised of 10,000 microprojections/cm2, each 250 µm long. It was hypothesized the higher density array with shallower ID delivery may be superior to the Mantoux technique. To test this hypothesis, HPV VLPs without adjuvant were coated on the Nanopatch™, stability of the Nanopatch™ with unadjuvanted HPV VLPs were evaluated under accelerated conditions, skin delivery was verified using radiolabelled VLPs or FluoSpheres®, and the immune response and skin site reaction with the Nanopatch™ was evaluated in rhesus macaques. The immune response induced by Nanopatch™ administration, measured as HPV-specific binding antibodies, was similar to that induced using the Mantoux technique. It was also observed that a lower dose of unadjuvanted HPV VLPs delivered with the first-generation Nanopatch™ and applicator or Mantoux technique resulted in an immune response that was significantly lower compared to a higher-dose of alum adjuvanted HPV VLPs delivered IM in rhesus macaques. The study also indicated unadjuvanted HPV VLPs could be delivered with the first-generation Nanopatch™ and applicator to the skin in 15 s with a transfer efficiency of approximately 20%. This study is the first demonstration of patch administration in non-human primates with a vaccine composed of HPV VLPs.

Potent neutralizing antibodies elicited by dengue vaccine in rhesus macaque target diverse epitopes.

There is still no safe and effective vaccine against dengue virus infection. Epidemics of dengue virus infection are increasingly a threat to human health around the world. Antibodies generated in response to dengue infection have been shown to impact disease development and effectiveness of dengue vaccine. In this study, we investigated monoclonal antibody responses to an experimental dengue vaccine in rhesus macaques. Variable regions of both heavy chain (VH) and light chain (VL) were cloned from single antibody-secreting B cells. A total of 780 monoclonal antibodies (mAbs) composed of paired VH and VL were characterized. Results show that the vaccination induces mAbs with diverse germline sequences and a wide range of binding affinities. Six potent neutralizing mAbs were identified among 130 dengue envelope protein binders. Critical amino acids for each neutralizing antibody binding to the dengue envelope protein were identified by alanine scanning of mutant libraries. Diverse epitopes were identified, including epitopes on the lateral ridge of DIII, the I-III hinge, the bc loop adjacent to the fusion loop of DII, and the ß-strands and loops of DI. Significantly, one of the neutralizing mAbs has a previously unknown epitope in DII at the interface of the envelope and membrane protein and is capable of neutralizing all four dengue serotypes. Taken together, the results of this study not only provide preclinical validation for the tested experimental vaccine, but also shed light on a potential application of the rhesus macaque model for better dengue vaccine evaluation and design of vaccines and immunization strategies.

Antibody responses to crucial functional epitopes as a novel approach to assess immunogenicity of vaccine adjuvants.

We are interested in developing a vaccine that prevents genital herpes. Adjuvants have a major impact on vaccine immunogenicity. We compared two adjuvants, an experimental Merck Sharp & Dohme lipid nanoparticle (LNP) adjuvant, LNP-2, with CpG oligonucleotide combined with alum for immunogenicity in mice when administered with herpes simplex virus type 2 (HSV-2) glycoproteins C, D and E (gC2, gD2, gE2). The immunogens are intended to produce neutralizing antibodies to gC2 and gD2, antibodies to gD2 and gE2 that block cell-to-cell spread, and antibodies to gE2 and gC2 that block immune evasion from antibody and complement, respectively. Overall, CpG/alum was better at producing serum and vaginal IgG binding antibodies, neutralizing antibodies, antibodies that block virus spread from cell-to-cell, and antibodies that block immune evasion domains on gC2. We used a novel high throughput biosensor assay to further assess differences in immunogenicity by mapping antibody responses to seven crucial epitopes on gD2 involved in virus entry or cell-to-cell spread. We found striking differences between CpG/alum and LNP-2. Mice immunized with gD2 CpG/alum produced higher titers of antibodies than LNP-2 to six of seven crucial epitopes and produced antibodies to more crucial epitopes than LNP-2. Measuring epitope-specific antibodies helped to define mechanisms by which CpG/alum outperformed LNP-2 and is a valuable technique to compare adjuvants.

Development and application of a higher throughput RSV plaque assay by immunofluorescent imaging.

Viral plaque assays are important tools in the development and evaluation of new antiviral drugs or vaccines in both preclinical and clinical research. While plaque assays are the standard tools to measure infectious virus, the methodology is time-consuming and requires experience in recognizing plaques. The assays are also prone to variation among analysts due to plaque recognition and manual counting errors. Here we describe the development of two simplified plaque assays for measuring RSV virus titers and anti-RSV antibody neutralization titers using 96 well plate formats. First, we evaluated multiple parameters to build up a quantitative plaque assay to measure infectious RSV. We then optimized the assay conditions to assess the fundamental changes from the traditional plaque assay, which were elimination of overnight pre-seeding host cells and addition of a centrifugation step after viral infection of the cells. We designed DoE to refine four key parameters within one experiment for host cell density, host cell volume, viral inoculum volume, host cell and viral mixture incubation time to make this assay more robust. We have also adapted these conditions into a second assay, which was an automated plaque reduction neutralization assay (PRNT) to determine neutralization titers of anti-RSV antibodies. Both assays utilize immune fluorescence staining to detect viral plaques. The images of the immuno-stained wells are captured by the PerkinElmer EnSight instrument and show clear visualization of plaques harvesting on day 3. Software algorithm was specifically designed for automatic counting of these fluorescent "objects". The quantitative plaque assay provided titers of RSV similar to those obtained from the traditional plaque assay. The method has been successfully utilized to screen multiple vaccine candidates in viral shedding efficacy studies. The automated PRNT assay provided antibody neutralizing titers that matched with published data. This automated 96 well plaque assay has made it possible to screen RSV samples in a higher throughput manner, and can be extended to other infectious organisms that form plaques for vaccine or drug evaluation.

Design and characterization of a fusion glycoprotein vaccine for Respiratory Syncytial Virus with improved stability.

Respiratory Syncytial Virus (RSV) infection is the leading cause of lower respiratory tract infection in both young children and older adults. Currently, there is no licensed vaccine available, and therapeutic options are limited. The infectious RSV particle is decorated with a type I viral fusion (F) glycoprotein that structurally rearranges from a metastable prefusion form to a highly stable postfusion form. In people naturally infected with RSV, the neutralizing antibodies primarily recognize the prefusion conformation. Therefore, engineered RSV F protein stabilized in its prefusion conformation has been an attractive strategy for developing RSV F vaccine antigens. Long-term stability at 4 °C or higher is a desirable attribute for a RSV F subunit vaccine antigen. We have previously shown that a prefusion stabilized RSV F construct, DS-Cav1, undergoes conformational changes and forms intermediate structures upon long-term storage at 4 °C. Structure-based design was performed to improve the stability of the RSV F subunit vaccine. We identified additional mutations that further stabilize RSV F protein in its prefusion conformation by using binding to a previously described antigenic site I antibody 4D7 as the screening tool. In addition, we designed and identified variants with increased expression levels, which is another desirable attribute for a subunit vaccine. Our data suggested that an RSV F variant F111 is properly folded, and has improved heat stability as well as stability upon long-term storage at 4 °C. A mouse immunogenicity study demonstrated that no compromise in immunogenicity (both binding and neutralizing antibody levels) was observed with the introduction of these additional mutations.

Co-Administration of Lipid Nanoparticles and Sub-Unit Vaccine Antigens Is Required for Increase in Antigen-Specific Immune Responses in Mice.

A vast body of evidence suggests that nanoparticles function as potent immune-modulatory agents. We have previously shown that Merck proprietary Lipid NanoParticles (LNPs) markedly boost B-cell and T-cell responses to sub-unit vaccine antigens in mice. To further evaluate the specifics of vaccine delivery and dosing regimens in vivo, we performed immunogenicity studies in BALB/c and C57BL/6 mice using two model antigens, Hepatitis B Surface Antigen (HBsAg) and Ovalbumin (OVA), respectively. To assess the requirement for co-administration of antigen and LNP for the elicitation of immune responses, we evaluated immune responses after administering antigen and LNP to separate limbs, or administering antigen and LNP to the same limb but separated by 24 h. We also evaluated formulations combining antigen, LNP, and aluminum-based adjuvant amorphous aluminum hydroxylphosphate sulfate (MAA) to look for synergistic adjuvant effects. Analyses of antigen-specific B-cell and T-cell responses from immunized mice revealed that the LNPs and antigens must be co-administered-both at the same time and in the same location-in order to boost antigen-specific immune responses. Mixing of antigen with MAA prior to formulation with LNP did not impact the generation of antigen-specific B-cell responses, but drastically reduced the ability of LNPs to boost antigen-specific T-cell responses. Overall, our data demonstrate that the administration of LNPs and vaccine antigen together enables their immune-stimulatory properties.

IMPre: An Accurate and Efficient Software for Prediction of T- and B-Cell Receptor Germline Genes and Alleles from Rearranged Repertoire Data.

Large-scale study of the properties of T-cell receptor (TCR) and B-cell receptor (BCR) repertoires through next-generation sequencing is providing excellent insights into the understanding of adaptive immune responses. Variable(Diversity)Joining [V(D)J] germline genes and alleles must be characterized in detail to facilitate repertoire analyses. However, most species do not have well-characterized TCR/BCR germline genes because of their high homology. Also, more germline alleles are required for humans and other species, which limits the capacity for studying immune repertoires. Herein, we developed "Immune Germline Prediction" (IMPre), a tool for predicting germline V/J genes and alleles using deep-sequencing data derived from TCR/BCR repertoires. We developed a new algorithm, "Seed_Clust," for clustering, produced a multiway tree for assembly and optimized the sequence according to the characteristics of rearrangement. We trained IMPre on human samples of T-cell receptor beta (TRB) and immunoglobulin heavy chain and then tested it on additional human samples. Accuracy of 97.7, 100, 92.9, and 100% was obtained for TRBV, TRBJ, IGHV, and IGHJ, respectively. Analyses of subsampling performance for these samples showed IMPre to be robust using different data quantities. Subsequently, IMPre was tested on samples from rhesus monkeys and human long sequences: the highly accurate results demonstrated IMPre to be stable with animal and multiple data types. With rapid accumulation of high-throughput sequence data for TCR and BCR repertoires, IMPre can be applied broadly for obtaining novel genes and a large number of novel alleles. IMPre is available at https://github.com/zhangwei2015/IMPre.

Stability Characterization of a Vaccine Antigen Based on the Respiratory Syncytial Virus Fusion Glycoprotein.

Infection with Respiratory Syncytial Virus (RSV) causes both upper and lower respiratory tract disease in humans, leading to significant morbidity and mortality in both young children and older adults. Currently, there is no licensed vaccine available, and therapeutic options are limited. During the infection process, the type I viral fusion (F) glycoprotein on the surface of the RSV particle rearranges from a metastable prefusion conformation to a highly stable postfusion form. In people naturally infected with RSV, most potent neutralizing antibodies are directed to the prefusion form of the F protein. Therefore, an engineered RSV F protein stabilized in the prefusion conformation (DS-Cav1) is an attractive vaccine candidate. Long-term stability at 4°C or higher is a desirable attribute for a commercial subunit vaccine antigen. To assess the stability of DS-Cav1, we developed assays using D25, an antibody which recognizes the prefusion F-specific antigenic site Ø, and a novel antibody 4D7, which was found to bind antigenic site I on the postfusion form of RSV F. Biophysical analysis indicated that, upon long-term storage at 4°C, DS-Cav1 undergoes a conformational change, adopting alternate structures that concomitantly lose the site Ø epitope and gain the ability to bind 4D7.

A Tetravalent Sub-unit Dengue Vaccine Formulated with Ionizable Cationic Lipid Nanoparticle induces Significant Immune Responses in Rodents and Non-Human Primates.

Dengue virus has emerged as an important arboviral infection worldwide. As a complex pathogen, with four distinct serotypes, the development of a successful Dengue virus vaccine has proven to be challenging. Here, we describe a novel Dengue vaccine candidate that contains truncated, recombinant, Dengue virus envelope protein from all four Dengue virus serotypes (DEN-80E) formulated with ionizable cationic lipid nanoparticles (LNPs). Immunization studies in mice, Guinea pigs, and in Rhesus macaques, revealed that LNPs induced high titers of Dengue virus neutralizing antibodies, with or without co-administration or encapsulation of a Toll-Like Receptor 9 agonist. Importantly, LNPs were also able to boost DEN-80E specific CD4+ and CD8+ T cell responses. Cytokine and chemokine profiling revealed that LNPs induced strong chemokine responses without significant induction of inflammatory cytokines. In addition to being highly efficacious, the vaccine formulation proved to be well-tolerated, demonstrating no elevation in any of the safety parameters evaluated. Notably, reduction in cationic lipid content of the nanoparticle dramatically reduced the LNP's ability to boost DEN-80E specific immune responses, highlighting the crucial role for the charge of the LNP. Overall, our novel studies, across multiple species, reveal a promising tetravalent Dengue virus sub-unit vaccine candidate.

A Rapid and Improved Method to Generate Recombinant Dengue Virus Vaccine Candidates.

Dengue is one of the most important mosquito-borne infections accounting for severe morbidity and mortality worldwide. Recently, the tetravalent chimeric live attenuated Dengue vaccine Dengvaxia® was approved for use in several dengue endemic countries. In general, live attenuated vaccines (LAV) are very efficacious and offer long-lasting immunity against virus-induced disease. Rationally designed LAVs can be generated through reverse genetics technology, a method of generating infectious recombinant viruses from full length cDNA contained in bacterial plasmids. In vitro transcribed (IVT) viral RNA from these infectious clones is transfected into susceptible cells to generate recombinant virus. However, the generation of full-length dengue virus cDNA clones can be difficult due to the genetic instability of viral sequences in bacterial plasmids. To circumvent the need for a single plasmid containing a full length cDNA, in vitro ligation of two or three cDNA fragments contained in separate plasmids can be used to generate a full-length dengue viral cDNA template. However, in vitro ligation of multiple fragments often yields low quality template for IVT reactions, resulting in inconsistent low yield RNA. These technical difficulties make recombinant virus recovery less efficient. In this study, we describe a simple, rapid and efficient method of using LONG-PCR to recover recombinant chimeric Yellow fever dengue (CYD) viruses as potential dengue vaccine candidates. Using this method, we were able to efficiently generate several viable recombinant viruses without introducing any artificial mutations into the viral genomes. We believe that the techniques reported here will enable rapid and efficient recovery of recombinant flaviviruses for evaluation as vaccine candidates and, be applicable to the recovery of other RNA viruses.

A novel lipid nanoparticle adjuvant significantly enhances B cell and T cell responses to sub-unit vaccine antigens.

Sub-unit vaccines are primarily designed to include antigens required to elicit protective immune responses and to be safer than whole-inactivated or live-attenuated vaccines. But their purity and inability to self-adjuvant often result in weaker immunogenicity. Emerging evidence suggests that bio-engineered nanoparticles can be used as immunomodulatory adjuvants. Therefore, in this study we explored the potential of novel Merck-proprietary lipid nanoparticle (LNP) formulations to enhance immune responses to sub-unit viral antigens. Immunization of BALB/c and C57BL/6 mice revealed that LNPs alone or in combination with a synthetic TLR9 agonist, immune-modulatory oligonucleotides, IMO-2125 (IMO), significantly enhanced immune responses to hepatitis B virus surface antigen (HBsAg) and ovalbumin (OVA). LNPs enhanced total B-cell responses to both antigens tested, to levels comparable to known vaccine adjuvants including aluminum based adjuvant, IMO alone and a TLR4 agonist, 3-O-deactytaled monophosphoryl lipid A (MPL). Investigation of the quality of B-cell responses demonstrated that the combination of LNP with IMO agonist elicited a stronger Th1-type response (based on the IgG2a:IgG1 ratio) than levels achieved with IMO alone. Furthermore, the LNP adjuvant significantly enhanced antigen specific cell-mediated immune responses. In ELISPOT assays, depletion of specific subsets of T cells revealed that the LNPs elicited potent antigen-specific CD4(+) and CD8(+)T cell responses. Intracellular FACS analyses revealed that LNP and LNP+IMO formulated antigens led to higher frequency of antigen-specific IFNγ(+)TNFα(+)IL-2(+), multi-functional CD8(+)T cell responses, than unadjuvanted vaccine or vaccine with IMO only. Overall, our results demonstrate that lipid nanoparticles can serve as future sub-unit vaccine adjuvants to boost both B-cell and T-cell responses in vivo, and that addition of IMO can be used to manipulate the quality of immune responses.

Preclinical and clinical development of a dengue recombinant subunit vaccine.

This review focuses on a dengue virus (DENV) vaccine candidate based on a recombinant subunit approach which targets the DENV envelope glycoprotein (E). Truncated versions of E consisting of the N-terminal portion of E (DEN-80E) have been expressed recombinantly in the Drosophila S2 expression system and shown to have native-like conformation. Preclinical studies demonstrate that formulations containing tetravalent DEN-80E adjuvanted with ISCOMATRIX™ adjuvant induce high titer virus neutralizing antibodies and IFN-γ producing T cells in flavivirus-naïve non-human primates. The preclinical data further suggest that administration of such formulations on a 0, 1, 6 month schedule may result in higher maximum virus neutralizing antibody titers and better durability of those titers compared to administration on a 0, 1, 2 month schedule. In addition, the virus neutralizing antibody titers induced by adjuvanted tetravalent DEN-80E compare favorably to the titers induced by a tetravalent live virus comparator. Furthermore, DEN-80E was demonstrated to be able to boost virus neutralizing antibody titers in macaques that have had a prior DENV exposure. A monovalent version of the vaccine candidate, DEN1-80E, was formulated with Alhydrogel™ and studied in a proof-of-principle Phase I clinical trial by Hawaii Biotech, Inc. (NCT00936429). The clinical trial results demonstrate that both the 10 µg and 50 µg formulations of DEN1-80E with 1.25 mg of elemental aluminum were immunogenic when administered in a 3-injection series (0, 1, 2 months) to healthy, flavivirus-naïve adults. The vaccine formulations induced DENV-1 neutralizing antibodies in the majority of subjects, although the titers in most subjects were modest and waned over time. Both the 10 µg DEN1-80E and the 50 µg DEN1-80E formulations with Alhydrogel™ were generally well tolerated.

Preclinical development of a dengue tetravalent recombinant subunit vaccine: Immunogenicity and protective efficacy in nonhuman primates.

We describe here the preclinical development of a dengue vaccine composed of recombinant subunit carboxy-truncated envelope (E) proteins (DEN-80E) for each of the four dengue serotypes. Immunogenicity and protective efficacy studies in Rhesus monkeys were conducted to evaluate monovalent and tetravalent DEN-80E vaccines formulated with ISCOMATRIX™ adjuvant. Three different doses and two dosing regimens (0, 1, 2 months and 0, 1, 2, and 6 months) were evaluated in these studies. We first evaluated monomeric (DEN4-80E) and dimeric (DEN4-80EZip) versions of DEN4-80E, the latter generated in an attempt to improve immunogenicity. The two antigens, evaluated at 6, 20 and 100 µg/dose formulated with ISCOMATRIX™ adjuvant, were equally immunogenic. A group immunized with 20 µg DEN4-80E and Alhydrogel™ induced much weaker responses. When challenged with wild-type dengue type 4 virus, all animals in the 6 and 20 µg groups and all but one in the DEN4-80EZip 100 µg group were protected from viremia. Two out of three monkeys in the Alhydrogel™ group had breakthrough viremia. A similar study was conducted to evaluate tetravalent formulations at low (3, 3, 3, 6 µg of DEN1-80E, DEN2-80E, DEN3-80E and DEN4-80E respectively), medium (10, 10, 10, 20 µg) and high (50, 50, 50, 100 µg) doses. All doses were comparably immunogenic and induced high titer, balanced neutralizing antibodies against all four DENV. Upon challenge with the four wild-type DENV, all animals in the low and medium dose groups were protected against viremia while two animals in the high-dose group exhibited breakthrough viremia. Our studies also indicated that a 0, 1, 2 and 6 month vaccination schedule is superior to the 0, 1, and 2 month schedule in terms of durability. Overall, the subunit vaccine was demonstrated to induce strong neutralization titers resulting in protection against viremia following challenge even 8-12 months after the last vaccine dose.