Route of self-amplifying mRNA vaccination modulates the establishment of pulmonary resident memory CD8 and CD4 T cells.

Respiratory tract resident memory T cells (TRM), typically generated by local vaccination or infection, can accelerate control of pulmonary infections that evade neutralizing antibody. It is unknown whether mRNA vaccination establishes respiratory TRM. We generated a self-amplifying mRNA vaccine encoding the influenza A virus nucleoprotein that is encapsulated in modified dendron-based nanoparticles. Here, we report how routes of immunization in mice, including contralateral versus ipsilateral intramuscular boosts, or intravenous and intranasal routes, influenced influenza-specific cell-mediated and humoral immunity. Parabiotic surgeries revealed that intramuscular immunization was sufficient to establish CD8 TRM in the lung and draining lymph nodes. Contralateral, compared with ipsilateral, intramuscular boosting broadened the distribution of lymph node TRM and T follicular helper cells but slightly diminished resulting levels of serum antibody. Intranasal mRNA delivery established modest circulating CD8 and CD4 T cell memory but augmented distribution to the respiratory mucosa. Combining intramuscular immunizations with an intranasal mRNA boost achieved high levels of both circulating T cell memory and lung TRM. Thus, routes of mRNA vaccination influence humoral and cell-mediated immunity, and intramuscular prime-boosting establishes lung TRM that can be further expanded by an additional intranasal immunization.

Antibody effector analysis of prime versus prime-boost immunizations with a recombinant measles-vectored chikungunya virus vaccine.

Chikungunya is a mosquito-borne disease that causes periodic but explosive epidemics of acute disease throughout the tropical world. Vaccine development against chikungunya virus (CHIKV) has been hampered by an inability to conduct efficacy trials due to the unpredictability of CHIKV outbreaks. Therefore, immune correlates are being explored to gain inference into vaccine-induced protection. This study is an in-depth serological characterization of Fab- and Fc-mediated antibody responses in selected phase II clinical trial participants following immunization with the recombinant measles-vectored CHIKV vaccine, MV-CHIK. Antibody comparisons were conducted between participants who received prime and those who received prime-boost vaccine regimens. MV-CHIK vaccination elicited potent Fab-mediated antibody responses (such as CHIKV-specific IgG, neutralization, and avidity), including dominant IgG3 responses, which translated into strong antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. At 1 month, prime-boost immunization led to significantly greater responses in every measured Fab and Fc antibody parameter. Interestingly, prime-boost-elicited antibodies decreased rapidly over time, until at 6 months both vaccine regimens displayed similar antibody profiles. Nonetheless, antibody avidity and antibody-dependent cellular phagocytosis remained significantly greater following boost immunization. Our observations suggest that a prime-boost administration of MV-CHIK will be more appropriate for CHIKV-endemic regions, while a prime-only regimen may be sufficient for travel purposes or outbreak situations.

Self-amplifying mRNA vaccines.

This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization.

A cationic nanoemulsion for the delivery of next-generation RNA vaccines.

Nucleic acid-based vaccines such as viral vectors, plasmid DNA, and mRNA are being developed as a means to address a number of unmet medical needs that current vaccine technologies have been unable to address. Here, we describe a cationic nanoemulsion (CNE) delivery system developed to deliver a self-amplifying mRNA vaccine. This nonviral delivery system is based on Novartis's proprietary adjuvant MF59, which has an established clinical safety profile and is well tolerated in children, adults, and the elderly. We show that nonviral delivery of a 9 kb self-amplifying mRNA elicits potent immune responses in mice, rats, rabbits, and nonhuman primates comparable to a viral delivery technology, and demonstrate that, relatively low doses (75 µg) induce antibody and T-cell responses in primates. We also show the CNE-delivered self-amplifying mRNA enhances the local immune environment through recruitment of immune cells similar to an MF59 adjuvanted subunit vaccine. Lastly, we show that the site of protein expression within the muscle and magnitude of protein expression is similar to a viral vector. Given the demonstration that self-amplifying mRNA delivered using a CNE is well tolerated and immunogenic in a variety of animal models, we are optimistic about the prospects for this technology.

Generation of a parvovirus B19 vaccine candidate.

Parvovirus B19 is the causative agent of fifth disease in children, aplastic crisis in those with blood dyscrasias, and hydrops fetalis. Previous parvovirus B19 virus-like-particle (VLP) vaccine candidates were produced by co-infection of insect cells with two baculoviruses, one expressing wild-type VP1 and the other expressing VP2. In humans, the VLPs were immunogenic but reactogenic. We have developed new VLP-based parvovirus B19 vaccine candidates, produced by co-expressing VP2 and either wild-type VP1 or phospholipase-negative VP1 in a regulated ratio from a single plasmid in Saccharomyces cerevisiae. These VLPs are expressed efficiently, are very homogeneous, and can be highly purified. Although VP2 alone can form VLPs, in mouse immunizations, VP1 and the adjuvant MF59 are required to elicit a neutralizing response. Wild-type VLPs and those with phospholipase-negative VP1 are equivalently potent. The purity, homogeneity, yeast origin, and lack of phospholipase activity of these VLPs address potential causes of previously observed reactogenicity.

RNA: the new revolution in nucleic acid vaccines.

Nucleic acid vaccines have the potential to address issues of safety and effectiveness sometimes associated with vaccines based on live attenuated viruses and recombinant viral vectors. In addition, methods to manufacture nucleic acid vaccines are suitable as generic platforms and for rapid response, both of which will be very important for addressing newly emerging pathogens in a timely fashion. Plasmid DNA is the more widely studied form of nucleic acid vaccine and proof of principle in humans has been demonstrated, although no licensed human products have yet emerged. The RNA vaccine approach, based on mRNA and engineered RNA replicons derived from certain RNA viruses, is gaining increased attention and several vaccines are under investigation for infectious diseases, cancer and allergy. Human clinical trials are underway and the prospects for success are bright.

Vectored co-delivery of human cytomegalovirus gH and gL proteins elicits potent complement-independent neutralizing antibodies.

Human cytomegalovirus (hCMV) is prevalent worldwide with infection generally being asymptomatic. Nevertheless, hCMV infection can lead to significant morbidity and mortality. Primary infection of seronegative women or reactivation/re-infection of seropositive women during pregnancy can result in transmission to the fetus, leading to severe neurological defects. In addition, hCMV is the most common viral infection in immunosuppressed organ transplant recipients and can produce serious complications. Hence, a safe and effective vaccine to prevent hCMV infection is an unmet medical need. Neutralizing antibodies to several hCMV glycoproteins, and complexes thereof, have been identified in individuals following hCMV infection. Interestingly, a portion of the CMV-specific neutralizing antibody responses are directed to epitopes found on glycoprotein complexes but not the individual proteins. Using an alphavirus replicon particle (VRP) vaccine platform, we showed that bicistronic VRPs encoding hCMV gH and gL glycoproteins produce gH/gL complexes in vitro. Furthermore, mice vaccinated with these gH/gL-expressing VRPs produced broadly cross-reactive complement-independent neutralizing antibodies to hCMV. These neutralizing antibody responses were of higher titer than those elicited in mice vaccinated with monocistronic VRPs encoding gH or gL antigens, and they were substantially more potent than those raised by VRPs encoding gB. These findings underscore the utility of co-delivery of glycoprotein components such as gH and gL for eliciting potent, broadly neutralizing immune responses against hCMV, and indicate that the gH/gL complex represents a potential target for future hCMV vaccine development.

Rapidly produced SAM(®) vaccine against H7N9 influenza is immunogenic in mice.

The timing of vaccine availability is essential for an effective response to pandemic influenza. In 2009, vaccine became available after the disease peak, and this has motivated the development of next generation vaccine technologies for more rapid responses. The SAM(®) vaccine platform, now in pre-clinical development, is based on a synthetic, self-amplifying mRNA, delivered by a synthetic lipid nanoparticle (LNP). When used to express seasonal influenza hemagglutinin (HA), a SAM vaccine elicited potent immune responses, comparable to those elicited by a licensed influenza subunit vaccine preparation. When the sequences coding for the HA and neuraminidase (NA) genes from the H7N9 influenza outbreak in China were posted on a web-based data sharing system, the combination of rapid and accurate cell-free gene synthesis and SAM vaccine technology allowed the generation of a vaccine candidate in 8 days. Two weeks after the first immunization, mice had measurable hemagglutinin inhibition (HI) and neutralizing antibody titers against the new virus. Two weeks after the second immunization, all mice had HI titers considered protective. If the SAM vaccine platform proves safe, potent, well tolerated and effective in humans, fully synthetic vaccine technologies could provide unparalleled speed of response to stem the initial wave of influenza outbreaks, allowing first availability of a vaccine candidate days after the discovery of a new virus.

Nonviral delivery of self-amplifying RNA vaccines.

Despite more than two decades of research and development on nucleic acid vaccines, there is still no commercial product for human use. Taking advantage of the recent innovations in systemic delivery of short interfering RNA (siRNA) using lipid nanoparticles (LNPs), we developed a self-amplifying RNA vaccine. Here we show that nonviral delivery of a 9-kb self-amplifying RNA encapsulated within an LNP substantially increased immunogenicity compared with delivery of unformulated RNA. This unique vaccine technology was found to elicit broad, potent, and protective immune responses, that were comparable to a viral delivery technology, but without the inherent limitations of viral vectors. Given the many positive attributes of nucleic acid vaccines, our results suggest that a comprehensive evaluation of nonviral technologies to deliver self-amplifying RNA vaccines is warranted.

RNA-based vaccines.

Nucleic acid vaccines consisting of plasmid DNA, viral vectors or RNA may change the way the next generation vaccines are produced, as they have the potential to combine the benefits of live-attenuated vaccines, without the complications often associated with live-attenuated vaccine safety and manufacturing. Over the past two decades, numerous clinical trials of plasmid DNA and viral vector-based vaccines have shown them to be safe, well-tolerated and immunogenic. Yet, sufficient potency for general utility in humans has remained elusive for DNA vaccines and the feasibility of repeated use of viral vectors has been compromised by anti-vector immunity. RNA vaccines, including those based on mRNA and self-amplifying RNA replicons, have the potential to overcome the limitations of plasmid DNA and viral vectors. Possible drawbacks related to the cost and feasibility of manufacturing RNA vaccines are being addressed, increasing the likelihood that RNA-based vaccines will be commercially viable. Proof of concept for RNA vaccines has been demonstrated in humans and the prospects for further development into commercial products are very encouraging.

Generation and genetic stability of tick-borne encephalitis virus mutants dependent on processing by the foot-and-mouth disease virus 3C protease.

Mature protein C of tick-borne encephalitis virus (TBEV) is cleaved from the polyprotein precursor by the viral NS2B/3 protease (NS2B/3(pro)). We showed previously that replacement of the NS2B/3(pro) cleavage site at the C terminus of protein C by the foot-and-mouth disease virus (FMDV) 2A StopGo sequence leads to the production of infectious virions. Here, we show that infectious virions can also be produced from a TBEV mutant bearing an inactivated 2A sequence through the expression of the FMDV 3C protease (3C(pro)) either in cis or in trans (from a TBEV replicon). Cleavage at the C terminus of protein C depended on the catalytic activity of 3C(pro) as well as on the presence of an optimized 3C(pro) cleavage site. Passage of the TBEV mutants bearing a 3C(pro) cleavage site either in the absence of 3C(pro) or in the presence of a catalytically inactive 3C(pro) led to the appearance of revertants in which protein C cleavage by NS2B/3(pro) had been regained. In three different revertants, a cleavage site for NS2B/3(pro), namely RR*C, was now present, leading to an elongated protein C. Furthermore, two revertants acquired additional mutations in the C terminus of protein C, eliminating two basic residues. Although these latter mutants showed wild-type levels of early RNA synthesis, their foci were smaller and an accumulation of protein C in the cytoplasm was observed. These findings suggest a role of the positive charge of the C terminus of protein C for budding of the nucleocapsid and further support the notion that TBEV protein C is a multifunctional protein.

Vaccines for the twenty-first century society.

Vaccines have been one of the major revolutions in the history of mankind and, during the twentieth century, they eliminated most of the childhood diseases that used to cause millions of deaths. In the twenty-first century, vaccines will also play a major part in safeguarding people's health. Supported by the innovations derived from new technologies, vaccines will address the new needs of a twenty-first century society characterized by increased life expectancy, emerging infections and poverty in low-income countries.

Mutational analysis of three predicted 5'-proximal stem-loop structures in the genome of tick-borne encephalitis virus indicates different roles in RNA replication and translation.

Flavivirus gene expression is modulated by RNA secondary structure elements at the terminal ends of the viral RNA molecule. For tick-borne encephalitis virus (TBEV), four stem-loop (SL) elements have been predicted in the first 180 nucleotides of the viral genome: 5'-SL1, 5'-SL2, 5'-SL3 and 5'-SL4. The last three of these appear to be unique to tick-borne flaviviruses. Here, we report their characterization by mutagenesis in a TBEV luciferase reporter system. By manipulating their thermodynamic properties, we found that an optimal stability of the 5'-SL2 is required for efficient RNA replication. 5'-SL3 formation is also important for viral RNA replication, but although it contains the viral start codon, its formation is dispensable for RNA translation. 5'-SL4 appears to facilitate both RNA translation and replication. Our data suggest that maintenance of the balanced thermodynamic stability of these SL elements is important for temporal regulation of its different functions.

Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers.

Respiratory syncytial virus (RSV), the main cause of infant bronchiolitis, remains a major unmet vaccine need despite more than 40 years of vaccine research. Vaccine candidates based on a chief RSV neutralization antigen, the fusion (F) glycoprotein, have foundered due to problems with stability, purity, reproducibility, and potency. Crystal structures of related parainfluenza F glycoproteins have revealed a large conformational change between the prefusion and postfusion states, suggesting that postfusion F antigens might not efficiently elicit neutralizing antibodies. We have generated a homogeneous, stable, and reproducible postfusion RSV F immunogen that elicits high titers of neutralizing antibodies in immunized animals. The 3.2-Å X-ray crystal structure of this substantially complete RSV F reveals important differences from homology-based structural models. Specifically, the RSV F crystal structure demonstrates the exposure of key neutralizing antibody binding sites on the surface of the postfusion RSV F trimer. This unanticipated structural feature explains the engineered RSV F antigen's efficiency as an immunogen. This work illustrates how structural-based antigen design can guide the rational optimization of candidate vaccine antigens.

Functional microRNA generated from a cytoplasmic RNA virus.

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that play a pivotal role in the regulation of posttranscriptional gene expression in a wide range of eukaryotic organisms. Although DNA viruses have been shown to encode miRNAs and exploit the cellular RNA silencing machinery as a convenient way to regulate viral and host gene expression, it is generally believed that this pathway is not available to RNA viruses that replicate in the cytoplasm of the cell because miRNA biogenesis is initiated in the nucleus. In fact, among the >200 viral miRNAs that have been experimentally verified so far, none is derived from an RNA virus. Here, we show that a cytoplasmic RNA virus can indeed encode and produce a functional miRNA. We introduced a heterologous miRNA-precursor stem-loop sequence element into the RNA genome of the flavivirus tick-borne encephalitis virus, and this led to the production of a functional miRNA during viral infection without impairing viral RNA replication. These findings demonstrate that miRNA biogenesis can be used by cytoplasmic RNA viruses to produce regulatory molecules for the modulation of the transcriptome.

Mutations at positions 186 and 194 in the HA gene of the 2009 H1N1 pandemic influenza virus improve replication in cell culture and eggs.

Obtaining suitable seed viruses for influenza vaccines poses a challenge for public health authorities and manufacturers. We used reverse genetics to generate vaccine seed-compatible viruses from the 2009 pandemic swine-origin influenza virus. Comparison of viruses recovered with variations in residues 186 and 194 (based on the H3 numbering system) of the viral hemagglutinin showed that these viruses differed with respect to their ability to grow in eggs and cultured cells. Thus, we have demonstrated that molecular cloning of members of a quasispecies can help in selection of seed viruses for vaccine manufacture.

Characterization of West Nile virus live vaccine candidates attenuated by capsid deletion mutations.

Protein C deletion mutants of West Nile virus (WNV) were evaluated for their potential use as live virus vaccine candidates in vivo. Double and triple mutants carrying small deletions and second-site point mutations, as well as mutants with large deletions of 36 and 37 amino acid residues were tested in a stringent mouse challenge model. The mutant viruses were found to be non-pathogenic and to induce protective immunity in a wide range of inoculation doses (10(1)-10(6)FFU). Furthermore, the effects of combining three different previously identified resuscitating point mutations, as well as the combination of a large protein C deletion with a deletion mutation in the 3' non-coding region were studied. The data indicate that the production of safe and efficacious WNV live vaccines based on protein C deletion mutations is feasible.

A trans-complementing recombination trap demonstrates a low propensity of flaviviruses for intermolecular recombination.

Intermolecular recombination between the genomes of closely related RNA viruses can result in the emergence of novel strains with altered pathogenic potential and antigenicity. Although recombination between flavivirus genomes has never been demonstrated experimentally, the potential risk of generating undesirable recombinants has nevertheless been a matter of concern and controversy with respect to the development of live flavivirus vaccines. As an experimental system for investigating the ability of flavivirus genomes to recombine, we developed a "recombination trap," which was designed to allow the products of rare recombination events to be selected and amplified. To do this, we established reciprocal packaging systems consisting of pairs of self-replicating subgenomic RNAs (replicons) derived from tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) that could complement each other in trans and thus be propagated together in cell culture over multiple passages. Any infectious viruses with intact, full-length genomes that were generated by recombination of the two replicons would be selected and enriched by end point dilution passage, as was demonstrated in a spiking experiment in which a small amount of wild-type virus was mixed with the packaged replicons. Using the recombination trap and the JEV system, we detected two aberrant recombination events, both of which yielded unnatural genomes containing duplications. Infectious clones of both of these genomes yielded viruses with impaired growth properties. Despite the fact that the replicon pairs shared approximately 600 nucleotides of identical sequence where a precise homologous crossover event would have yielded a wild-type genome, this was not observed in any of these systems, and the TBEV and WNV systems did not yield any viable recombinant genomes at all. Our results show that intergenomic recombination can occur in the structural region of flaviviruses but that its frequency appears to be very low and that therefore it probably does not represent a major risk in the use of live, attenuated flavivirus vaccines.

Extension of flavivirus protein C differentially affects early RNA synthesis and growth in mammalian and arthropod host cells.

The translation of flaviviral RNA genomes yields a single polyprotein that is processed into the mature proteins by viral and host cell proteases. Mature capsid protein C is freed from the polyprotein by the viral NS2B/3 protease, cleaving in the C-terminal region of protein C in front of the signal sequence for prM. Protein C has been shown to be involved in viral assembly and RNA packaging. To examine further the role of protein C and its production by proteolysis, we replaced the NS2B/3 capsid cleavage site in tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) by the 2A protein of foot-and-mouth disease virus (TBEV-2A and WNV-2A). This obviated the need for NS2B/3 processing at the C terminus of mature protein C while simultaneously producing a 19-amino-acid extension on protein C. Infectious virions were generated with both viruses; the phenotype depended on the host cell. TBEV-2A replicated well in BHK-21 cells but was essentially incapable of replication in tick cells. In contrast, WNV-2A replicated well in mosquito cells but showed a small-plaque phenotype in Vero cells, with frequent production of larger plaques. Sequencing of viral RNA from the larger plaques showed substitutions in the signal sequence for prM, presumably improving coordinated protein processing at the C-prM junction. Furthermore, both TBEV-2A and WNV-2A were also defective in unpackaging and/or early RNA synthesis. Together, these results indicate a role for flavivirus protein C in both viral assembly and RNA replication, possibly by interacting with host cell factors required to set up the cell for RNA replication.