Michael Boyd

Vaccine protection against Zika virus from Brazil

Zika virus (ZIKV) is a flavivirus that is responsible for the current epidemic in Brazil and the Americas. ZIKV has been causally associated with fetal microcephaly, intrauterine growth restriction, and other birth defects in both humans and mice. The rapid development of a safe and effective ZIKV vaccine is a global health priority, but very little is currently known about ZIKV immunology and mechanisms of immune protection. Here we show that a single immunization with a plasmid DNA vaccine or a purified inactivated virus vaccine provides complete protection in susceptible mice against challenge with a strain of ZIKV involved in the outbreak in northeast Brazil. This ZIKV strain has recently been shown to cross the placenta and to induce fetal microcephaly and other congenital malformations in mice. We produced DNA vaccines expressing ZIKV pre-membrane and envelope (prM-Env), as well as a series of deletion mutants. The prM-Env DNA vaccine, but not the deletion mutants, afforded complete protection against ZIKV, as measured by absence of detectable viraemia following challenge, and protective efficacy correlated with Env-specific antibody titers. Adoptive transfer of purified IgG from vaccinated mice conferred passive protection, and depletion of CD4 and CD8 T lymphocytes in vaccinated mice did not abrogate this protection. These data demonstrate that protection against ZIKV challenge can be achieved by single-shot subunit and inactivated virus vaccines in mice and that Env-specific antibody titers represent key immunologic correlates of protection. Our findings suggest that the development of a ZIKV vaccine for humans is likely to be achievable.

Protective efficacy of multiple vaccine platforms against Zika virus challenge in rhesus monkeys

Zika virus (ZIKV) is responsible for a major ongoing epidemic in the Americas and has been causally associated with fetal microcephaly. The development of a safe and effective ZIKV vaccine is therefore an urgent global health priority. Here we demonstrate that three different vaccine platforms protect against ZIKV challenge in rhesus monkeys. A purified inactivated virus vaccine induced ZIKV-specific neutralizing antibodies and completely protected monkeys against ZIKV strains from both Brazil and Puerto Rico. Purified immunoglobulin from vaccinated monkeys also conferred passive protection in adoptive transfer studies. A plasmid DNA vaccine and a single-shot recombinant rhesus adenovirus serotype 52 vector vaccine, both expressing ZIKV premembrane and envelope, also elicited neutralizing antibodies and completely protected monkeys against ZIKV challenge. These data support the rapid clinical development of ZIKV vaccines for humans.

Rapid development of a DNA vaccine for Zika virus

A DNA vaccine candidate for Zika The ongoing Zika epidemic in the Americas and the Caribbean urgently needs a protective vaccine. Two DNA vaccines composed of the genes that encode the structural premembrane and envelope proteins of Zika virus have been tested in monkeys. Dowd et al. show that two doses of vaccine given intramuscularly completely protected 17 of 18 animals against Zika virus challenge. A single low dose of vaccine was not protective but did reduce viral loads. Protection correlated with serum antibody neutralizing activity. Phase I clinical trials testing these vaccines are already ongoing. Science, this issue p. 237 DNA-vaccine–induced neutralizing antibodies largely protect monkeys after experimental challenge by virus infection. 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.

Lung-toxic furanoterpenoids produced by sweet potatoes (Ipomoea batatas) following microbial infection☆

Abstract The pulmonary toxicity of mold-damaged sweet potatoes has recently been ascribed to a “lung edema factor” which is produced in the roots in response to microbial infection. We now present data suggesting that this toxic principle comprises a group of at least four closely related compounds: 4-ipomeanol (1-(3-furyl)-4-hydroxy-1-pentanone), the isomeric 1-ipomeanol (1-(3-furyl)-1-hydroxy-4-pentanone). the corresponding diketone, ipomeanine (1-(3-furyl)-1,4-pentanedione), and the diol, 1,4-ipomeadiol (1-(3-furyl)-1,4-pentanediol). Isolation and purification of the compounds was accomplished using a combination of column chromatography, preparative gas chromatography, and high pressure liquid chromatography. Structures were initially determined from infrared, nuclear magnetic resonance, and mass spectra. Synthetic procedures for the compounds were devised in order to confirm the assigned structures and to provide an efficient source of the toxins for biological studies. All of these 1,4-dioxygenated-1-(3-furyl)-pentanes arc acutely toxic to the lungs of experimental animals, characteristically producing pulmonary edema and congestion, following a latent period of several hours after dosing. Mice receiving lethal doses of the toxins usually die within 24 h, and pathological findings are seen most often only in the lungs. However, mice initially surviving near-lethal doses of the toxins, particularly 1-ipomeanol and 1,4-ipomeadiol, may show evidence of nephrotoxicity within 1–3 days. The latter observation suggests that pathological responses other than lung damage should also be considered as possible features of moldy sweet potato toxicity.

Zika Virus Persistence in the Central Nervous System and Lymph Nodes of Rhesus Monkeys

Zika virus (ZIKV) is associated with severe neuropathology in neonates as well as Guillain-Barré syndrome and other neurologic disorders in adults. Prolonged viral shedding has been reported in semen, suggesting the presence of anatomic viral reservoirs. Here we show that ZIKV can persist in cerebrospinal fluid (CSF) and lymph nodes (LN) of infected rhesus monkeys for weeks after virus has been cleared from peripheral blood, urine, and mucosal secretions. ZIKV-specific neutralizing antibodies correlated with rapid clearance of virus in peripheral blood but remained undetectable in CSF for the duration of the study. Viral persistence in both CSF and LN correlated with upregulation of mechanistic target of rapamycin (mTOR), proinflammatory, and anti-apoptotic signaling pathways, as well as downregulation of extracellular matrix signaling pathways. These data raise the possibility that persistent or occult neurologic and lymphoid disease may occur following clearance of peripheral virus in ZIKV-infected individuals.

Impact of prior flavivirus immunity on Zika virus infection in rhesus macaques

Studies have demonstrated cross-reactivity of anti-dengue virus (DENV) antibodies in human sera against Zika virus (ZIKV), promoting increased ZIKV infection in vitro. However, the correlation between in vitro and in vivo findings is not well characterized. Thus, we evaluated the impact of heterotypic flavivirus immunity on ZIKV titers in biofluids of rhesus macaques. Animals previously infected (≥420 days) with DENV2, DENV4, or yellow fever virus were compared to flavivirus-naïve animals following infection with a Brazilian ZIKV strain. Sera from DENV-immune macaques demonstrated cross-reactivity with ZIKV by antibody-binding and neutralization assays prior to ZIKV infection, and promoted increased ZIKV infection in cell culture assays. Despite these findings, no significant differences between flavivirus-naïve and immune animals were observed in viral titers, neutralizing antibody levels, or immune cell kinetics following ZIKV infection. These results indicate that prior infection with heterologous flaviviruses neither conferred protection nor increased observed ZIKV titers in this non-human primate ZIKV infection model.

Preliminary aggregate safety and immunogenicity results from three trials of a purified inactivated Zika virus vaccine candidate: phase 1, randomised, double-blind, placebo-controlled clinical trials

BACKGROUND A safe, effective, and rapidly scalable vaccine against Zika virus infection is needed. We developed a purified formalin-inactivated Zika virus vaccine (ZPIV) candidate that showed protection in mice and non-human primates against viraemia after Zika virus challenge. Here we present the preliminary results in human beings. METHODS We did three phase 1, placebo-controlled, double-blind trials of ZPIV with aluminium hydroxide adjuvant. In all three studies, healthy adults were randomly assigned by a computer-generated list to receive 5 μg ZPIV or saline placebo, in a ratio of 4:1 at Walter Reed Army Institute of Research, Silver Spring, MD, USA, or of 5:1 at Saint Louis University, Saint Louis, MO, USA, and Beth Israel Deaconess Medical Center, Boston, MA, USA. Vaccinations were given intramuscularly on days 1 and 29. The primary objective was safety and immunogenicity of the ZPIV candidate. We recorded adverse events and Zika virus envelope microneutralisation titres up to day 57. These trials are registered at ClinicalTrials.gov, numbers NCT02963909, NCT02952833, and NCT02937233. FINDINGS We enrolled 68 participants between Nov 7, 2016, and Jan 25, 2017. One was excluded and 67 participants received two injections of Zika vaccine (n=55) or placebo (n=12). The vaccine caused only mild to moderate adverse events. The most frequent local effects were pain (n=40 [60%]) or tenderness (n=32 [47%]) at the injection site, and the most frequent systemic reactogenic events were fatigue (29 [43%]), headache (26 [39%]), and malaise (15 [22%]). By day 57, 52 (92%) of vaccine recipients had seroconverted (microneutralisation titre ≥1:10), with peak geometric mean titres seen at day 43 and exceeding protective thresholds seen in animal studies. INTERPRETATION The ZPIV candidate was well tolerated and elicited robust neutralising antibody titres in healthy adults. FUNDING Departments of the Army and Defense and National Institute of Allergy and Infectious Diseases.

Development of tolerance to the pulmonary toxin, 4-ipomeanol

Abstract Tolerance to the pulmonary toxin, 4-ipomeanol, administered i.p. to mice, rats, or rabbits, or given i.v. to rats, was induced rapidly by i.p. pretreatments with small doses of 4-ipomeanol itself. Tolerance was inducible to a similar extent in male or female rats. In mice, the degree of tolerance was somewhat dependent upon the particular pretreatment regimen used. Ipomeanine, a lung-toxic furan that is closely related in structure to 4-ipomeanol, showed a cross-tolerance phenomenon with 4-ipomeanol in mice, but analogs of 4-ipomeanol that did not contain a furan ring, and which were not lung-toxic, did not induce tolerance. Pretreatments of mice with 4-ipomeanol produced tolerance to another type of lung toxin, α-naphthylthiourea (ANTU), but ANTU pretreatments did not produce tolerance to 4-ipomeanol. Secondary pretreatments with CCl4 or SKF-525A modified the degree of tolerance to 4-ipomeanol in 4-ipomeanol-pretreated animals, but CCl4 pretreatment alone did not alter the lethality of 4-ipomeanol. Pretreatments of mice with phenobarbital or 3-methylcholanthrene decreased the lethality of 4-ipomeanol. However, pretreatments of mice with tolerance-inducing doses of 4-ipomeanol did not alter the hexobarbital sleeping times or the zoxazolamine paralysis times in mice, suggesting that tolerance due to phenobarbital or 3-methylcholanthrene pretreatments was by a different mechanism than that resulting from 4-ipomeanol pretreatment. The amounts of 14CO2 appearing in the expired air were the same in control and tolerant rats administered [2-14C]ethanol. On the other hand, the amounts of radiolabeled 4-ipomeanol metabolites bound covalently to the lungs were markedly lower in 4-ipomeanol pretreated rats than in control rats; this observation is consistent with previous studies indicating that potentially lethal pulmonary damage by 4-ipomeanol is caused by an alkylating metabolite. Tolerance to 4-ipomeanol apparently results from an alteration in metabolism of the compound along toxifying and/or detoxifying pathways, but further studies are needed to determine the relative importance of changes in the 2 types of pathways. The tolerance phenomenon demonstrated with 4-ipomeanol may provide a useful model for investigating tolerance mechanisms generally applicable to other lung-toxic chemical and gases.

Durability and correlates of vaccine protection against Zika virus in rhesus monkeys

Not all vaccines afford robust protection against ZIKV challenge in rhesus monkeys at 1 year after vaccination. Patience pays off As an individual may not encounter the pathogen for years after they have been vaccinated, efficacious vaccines typically require induction of long-lasting immunity. Abbink and colleagues vaccinated nonhuman primates with one of several candidate Zika virus vaccines and then waited an entire year before conducting a viral challenge. These vaccines had all shown promising results in previous experiments with a more immediate challenge, but here, one vaccine faltered, likely due to waning antibodies. The researchers performed more experiments to suggest that circulating antibodies are mediating protection for these vaccines. These results are useful for Zika virus vaccine development and instructive for vaccine development in general. An effective Zika virus (ZIKV) vaccine will require long-term durable protection. Several ZIKV vaccine candidates have demonstrated protective efficacy in nonhuman primates, but these studies have typically involved ZIKV challenge shortly after vaccination at peak immunity. We show that a single immunization with an adenovirus vector–based vaccine, as well as two immunizations with a purified inactivated virus vaccine, afforded robust protection against ZIKV challenge in rhesus monkeys at 1 year after vaccination. In contrast, two immunizations with an optimized DNA vaccine, which provided complete protection at peak immunity, resulted in reduced protective efficacy at 1 year that was associated with declining neutralizing antibody titers to subprotective levels. These data define a microneutralization log titer of 2.0 to 2.1 as the threshold required for durable protection against ZIKV challenge in this model. Moreover, our findings demonstrate that protection against ZIKV challenge in rhesus monkeys is possible for at least 1 year with a single-shot vaccine.

Production of Mucosally Transmissible SHIV Challenge Stocks from HIV-1 Circulating Recombinant Form 01_AE env Sequences

Simian-human immunodeficiency virus (SHIV) challenge stocks are critical for preclinical testing of vaccines, antibodies, and other interventions aimed to prevent HIV-1. A major unmet need for the field has been the lack of a SHIV challenge stock expressing circulating recombinant form 01_AE (CRF01_AE) env sequences. We therefore sought to develop mucosally transmissible SHIV challenge stocks containing HIV-1 CRF01_AE env derived from acutely HIV-1 infected individuals from Thailand. SHIV-AE6, SHIV-AE6RM, and SHIV-AE16 contained env sequences that were >99% identical to the original HIV-1 isolate and did not require in vivo passaging. These viruses exhibited CCR5 tropism and displayed a tier 2 neutralization phenotype. These challenge stocks efficiently infected rhesus monkeys by the intrarectal route, replicated to high levels during acute infection, and established chronic viremia in a subset of animals. SHIV-AE16 was titrated for use in single, high dose as well as repetitive, low dose intrarectal challenge studies. These SHIV challenge stocks should facilitate the preclinical evaluation of vaccines, monoclonal antibodies, and other interventions targeted at preventing HIV-1 CRF01_AE infection.