Development of a potent Zika virus vaccine using self-amplifying messenger RNA.

Zika virus (ZIKV) is the cause of a pandemic associated with microcephaly in newborns and Guillain-Barre syndrome in adults. Currently, there are no available treatments or vaccines for ZIKV, and the development of a safe and effective vaccine is a high priority for many global health organizations. We describe the development of ZIKV vaccine candidates using the self-amplifying messenger RNA (SAM) platform technology delivered by cationic nanoemulsion (CNE) that allows bedside mixing and is particularly useful for rapid responses to pandemic outbreaks. Two immunizations of either of the two lead SAM (CNE) vaccine candidates elicited potent neutralizing antibody responses to ZIKV in mice and nonhuman primates. Both SAM (CNE) vaccines protected these animals from ZIKV challenge, with one candidate providing complete protection against ZIKV infection in nonhuman primates. The data provide a preclinical proof of concept that a SAM (CNE) vaccine candidate can rapidly elicit protective immunity against ZIKV.

Distinct neutralizing antibody correlates of protection among related Zika virus vaccines identify a role for antibody quality.

The emergence of Zika virus (ZIKV) in the Americas stimulated the development of multiple ZIKV vaccine candidates. We previously developed two related DNA vaccine candidates encoding ZIKV structural proteins that were immunogenic in animal models and humans. We sought to identify neutralizing antibody (NAb) properties induced by each vaccine that correlated with protection in nonhuman primates (NHPs). Despite eliciting equivalent NAb titers in NHPs, these vaccines were not equally protective. The transfer of equivalent titers of vaccine-elicited NAb into AG129 mice also revealed nonequivalent protection, indicating qualitative differences among antibodies (Abs) elicited by these vaccines. Both vaccines elicited Abs with similar binding titers against envelope protein monomers and those incorporated into virus-like particles, as well as a comparable capacity to orchestrate phagocytosis. Functional analysis of vaccine-elicited NAbs from NHPs and humans revealed a capacity to neutralize the structurally mature form of the ZIKV virion that varied in magnitude among vaccine candidates. Conversely, sensitivity to the virion maturation state was not a characteristic of NAbs induced by natural or experimental infection. Passive transfer experiments in mice revealed that neutralization of mature ZIKV virions more accurately predicts protection from ZIKV infection. These findings demonstrate that NAb correlates of protection may differ among vaccine antigens when assayed using standard neutralization platforms and suggest that measurements of Ab quality, including the capacity to neutralize mature virions, will be critical for defining correlates of ZIKV vaccine-induced immunity.

The Zika virus envelope protein glycan loop regulates virion antigenicity.

Because antibodies are an important component of flavivirus immunity, understanding the antigenic structure of flaviviruses is critical. Compared to dengue virus (DENV), the loop containing the single N-linked glycosylation site on Zika virus (ZIKV) envelope (E) proteins extends further towards the DII fusion loop (DII-FL) on neighboring E proteins within E dimers on mature viruses. Although ZIKV is poorly neutralized by DII-FL antibodies, we demonstrated significantly increased neutralization sensitivity of ZIKV particles incorporating the DENV glycan loop. Increased neutralization sensitivity was independent of E protein glycosylation: ZIKV lacking E protein glycans remained poorly neutralized, whereas ZIKV loop chimeras with or without an E protein glycan were potently neutralized. ZIKV particles lacking the E protein glycan were capable of infecting Raji cells expressing the lectin DC-SIGNR, suggesting the prM glycan of partially mature particles can facilitate entry. Our study provides insight into the determinants of ZIKV E protein function and antigenicity.

Safety, tolerability, and immunogenicity of two Zika virus DNA vaccine candidates in healthy adults: randomised, open-label, phase 1 clinical trials.

BACKGROUND: The Zika virus epidemic and associated congenital infections have prompted rapid vaccine development. We assessed two new DNA vaccines expressing premembrane and envelope Zika virus structural proteins. METHODS: We did two phase 1, randomised, open-label trials involving healthy adult volunteers. The VRC 319 trial, done in three centres, assessed plasmid VRC5288 (Zika virus and Japanese encephalitis virus chimera), and the VRC 320, done in one centre, assessed plasmid VRC5283 (wild-type Zika virus). Eligible participants were aged 18-35 years in VRC19 and 18-50 years in VRC 320. Participants were randomly assigned 1:1 by a computer-generated randomisation schedule prepared by the study statistician. All participants received intramuscular injection of 4 mg vaccine. In VRC 319 participants were assigned to receive vaccinations via needle and syringe at 0 and 8 weeks, 0 and 12 weeks, 0, 4, and 8 weeks, or 0, 4, and 20 weeks. In VRC 320 participants were assigned to receive vaccinations at 0, 4, and 8 weeks via single-dose needle and syringe injection in one deltoid or split-dose needle and syringe or needle-free injection with the Stratis device (Pharmajet, Golden, CO, USA) in each deltoid. Both trials followed up volunteers for 24 months for the primary endpoint of safety, assessed as local and systemic reactogenicity in the 7 days after each vaccination and all adverse events in the 28 days after each vaccination. The secondary endpoint in both trials was immunogenicity 4 weeks after last vaccination. These trials are registered with ClinicalTrials.gov, numbers NCT02840487 and NCT02996461. FINDINGS: VRC 319 enrolled 80 participants (20 in each group), and VRC 320 enrolled 45 participants (15 in each group). One participant in VRC 319 and two in VRC 320 withdrew after one dose of vaccine, but were included in the safety analyses. Both vaccines were safe and well tolerated. All local and systemic symptoms were mild to moderate. In both studies, pain and tenderness at the injection site was the most frequent local symptoms (37 [46%] of 80 participants in VRC 319 and 36 [80%] of 45 in VRC 320) and malaise and headache were the most frequent systemic symptoms (22 [27%] and 18 [22%], respectively, in VRC 319 and 17 [38%] and 15 [33%], respectively, in VRC 320). For VRC5283, 14 of 14 (100%) participants who received split-dose vaccinations by needle-free injection had detectable positive antibody responses, and the geometric mean titre of 304 was the highest across all groups in both trials. INTERPRETATION: VRC5283 was well tolerated and has advanced to phase 2 efficacy testing. FUNDING: Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination.

Zika virus (ZIKV) has recently emerged as a pandemic associated with severe neuropathology in newborns and adults. There are no ZIKV-specific treatments or preventatives. Therefore, the development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins. Here we demonstrate that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) encoding the pre-membrane and envelope glycoproteins of a strain from the ZIKV outbreak in 2013 elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 µg of nucleoside-modified ZIKV mRNA-LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50 µg was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination. These data demonstrate that nucleoside-modified mRNA-LNP elicits rapid and durable protective immunity and therefore represents a new and promising vaccine candidate for the global fight against ZIKV.

Rapid development of a DNA vaccine for Zika virus.

Zika virus (ZIKV) was identified as a cause of congenital disease during the explosive outbreak in the Americas and Caribbean that began in 2015. Because of the ongoing fetal risk from endemic disease and travel-related exposures, a vaccine to prevent viremia in women of childbearing age and their partners is imperative. We found that vaccination with DNA expressing the premembrane and envelope proteins of ZIKV was immunogenic in mice and nonhuman primates, and protection against viremia after ZIKV challenge correlated with serum neutralizing activity. These data not only indicate that DNA vaccination could be a successful approach to protect against ZIKV infection, but also suggest a protective threshold of vaccine-induced neutralizing activity that prevents viremia after acute infection.

Zika Virus Is Not Uniquely Stable at Physiological Temperatures Compared to Other Flaviviruses.

UNLABELLED: Zika virus (ZIKV) is a flavivirus that has emerged as a global health threat due in part to its association with congenital abnormalities. Other globally relevant flaviviruses include dengue virus (DENV) and West Nile virus (WNV). High-resolution structures of ZIKV reveal many similarities to DENV and suggest some differences, including an extended glycan loop (D. Sirohi, Z. Chen, L. Sun, T. Klose, T. C. Pierson, et al., 352:467-470, 2016, http://dx.doi.org/10.1126/science.aaf5316) and unique interactions among envelope (E) protein residues that were proposed to confer increased virion stability and contribute mechanistically to the distinctive pathobiology of ZIKV (V. A. Kostyuchenko, E. X. Lim, S. Zhang, G. Fibriansah, T. S. Ng, et al., Nature 533:425-428, 2016, http://dx.doi.org/10.1038/nature17994). However, in the latter study, virus stability was inferred by measuring the loss of infectivity following a short incubation period. Here, we rigorously assessed the relative stability of ZIKV, DENV, and WNV by measuring changes in infectivity following prolonged incubation at physiological temperatures. At 37°C, the half-life of ZIKV was approximately twice as long as the half-life of DENV (11.8 and 5.2 h, respectively) but shorter than that of WNV (17.7 h). Incubation at 40°C accelerated the loss of ZIKV infectivity. Increasing virion maturation efficiency modestly increased ZIKV stability, as observed previously with WNV and DENV. Finally, mutations at E residues predicted to confer increased stability to ZIKV did not affect virion half-life. Our results demonstrate that ZIKV is not uniquely stable relative to other flaviviruses, suggesting that its unique pathobiology is explained by an alternative mechanism. IMPORTANCE: Zika virus (ZIKV) belongs to the Flavivirus genus, which includes other clinically relevant mosquito-borne pathogens such as dengue virus (DENV) and West Nile virus (WNV). Historically, ZIKV infection was characterized by a self-limiting, mild disease, but recent outbreaks have been associated with severe clinical complications, including Guillain-Barré syndrome and microcephaly, which are atypical of other flavivirus infections. Moreover, ZIKV has been detected in saliva, urine, and semen, and it may be sexually transmitted. Analysis of a high-resolution cryo-electron microscopic reconstruction of ZIKV hypothesized that the unusual stability of this virus contributes to its distinctive pathobiology. Here, we directly compared the stability of ZIKV to that of other flaviviruses following prolonged incubation in solution at physiological temperatures. We found that the stability of multiple ZIKV strains, including those from recent outbreaks, is intermediate between that of DENV and WNV, suggesting an alternative explanation for the unique clinical manifestations of ZIKV infection.

Norharmane Matrix Enhances Detection of Endotoxin by MALDI-MS for Simultaneous Profiling of Pathogen, Host, and Vector Systems.

The discovery of novel pathogenic mechanisms engaged during bacterial infections requires the evolution of advanced techniques. Here, we evaluate the dual polarity matrix norharmane (NRM) to improve detection of bacterial lipid A (endotoxin), from host and vector tissues infected with Francisella novicida (Fn). We evaluated NRM for improved detection and characterization of a wide range of lipids in both positive and negative polarities, including lipid A and phospholipids across a range of matrix assisted laser desorption-ionization (MALDI)-coupled applications. NRM matrix improved the limit of detection (LOD) for monophosphoryl lipid A (MPLA) down to picogram-level representing a ten-fold improvement of LOD versus 2,5-dihydroxybenzoic acid (DHB) and 100-fold improvement of LOD versus 9-aminoacridine (9-AA). Improved LOD for lipid A subsequently facilitated detection of the Fn lipid A major ion (m/z 1665) from extracts of infected mouse spleen and the temperature-modified Fn lipid A at m/z 1637 from infected D. variabilis ticks. Finally, we simultaneously mapped bacterial phospholipid signatures within an Fn infected spleen along with exclusively host-derived inositol-based phospholipid (m/z 933) demonstrating co-profiling for the host-pathogen interaction. Expanded use of NRM matrix in other infection models and endotoxin-targeting imaging experiments will improve our understanding of the lipid interactions at the host-pathogen interface.

Broadly Neutralizing Activity of Zika Virus-Immune Sera Identifies a Single Viral Serotype.

Recent epidemics of Zika virus (ZIKV) have been associated with congenital malformation during pregnancy and Guillain-Barré syndrome. There are two ZIKV lineages (African and Asian) that share >95% amino acid identity. Little is known regarding the ability of neutralizing antibodies elicited against one lineage to protect against the other. We investigated the breadth of the neutralizing antibody response following ZIKV infection by measuring the sensitivity of six ZIKV strains to neutralization by ZIKV-confirmed convalescent human serum or plasma samples. Contemporary Asian and early African ZIKV strains were similarly sensitive to neutralization regardless of the cellular source of virus. Furthermore, mouse immune serum generated after infection with African or Asian ZIKV strains was capable of neutralizing homologous and heterologous ZIKV strains equivalently. Because our study only defines a single ZIKV serotype, vaccine candidates eliciting robust neutralizing antibody responses should inhibit infection of both ZIKV lineages, including strains circulating in the Americas.

Disrupting protein expression with Peptide Nucleic Acids reduces infection by obligate intracellular Rickettsia.

Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria's ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria.