Weanling Swiss Webster mice were inoculated intraperitoneally with 100 LD50 of LACV/human/1960, and tissues were collected for six consecutive days for virus titration (n = 5) and pathology (n = 3) per day. Virus titrations were performed to confirm previous virus kinetics. Tissues collected for pathology were fixed in 10% neutral buffered formalin (NBF) for a minimum of 72 hours, embedded in paraffin and sections were prepared at 4–5 (μm). When bone was present in the tissue, as with muscle, nasal cavity, and spinal cord, tissues were decalcified using Immunocal (Decal Chemical Corp. Tallman, NY). Sections were stained with hematoxylin and eosin (H&E). A serial section was saved for immunohistochemical staining (see below).

For immunohistochemical analysis, fixed serial sections of mouse tissues were mounted onto slides and deparaffinized with xylene, rehydrated in a series of ethanol solutions (100%, 95%, 70%, 50%), and washed with deionized water. The sections were first treated with peroxidase blocking solution [2% H2O2(30%), 80% methanol, 18% dH2O] at room temperature for 5 minutes to quench endogenous peroxidases. Sections were pretreated with Pro K enzyme (Dako Corp., Carpinteria CA) at room temperature for 10 minutes and stained using the Mouse on Mouse Polymer detection system (Biocare Medical, Concord, CA). The primary antibody, mouse anti-La Crosse virus monoclonal antibody #18752 (QED Bioscience INC, San Diego, CA), recognized the G2 (Gn) glycoprotein and was used at a dilution of 1:200.

TUNEL Staining to detect apoptosis was preformed using the "DeadEnd™ Colorimetric TUNEL System" (Promega USA, Madison, WI). Sections were pretreated with Pro K enzyme (provided in the kit, diluted 1:500 with PBS). Strepavidin (also provided in the kit) was diluted at 1:500 in PBS.

To detect CD3+cells, slides were steam hydrated and pretreated with Diva Solution (Biocare Medical Concord, CA) for 20 minutes. The primary antibody, rabbit anti-human CD3, (A-0452, Dako Corporation, Carpentaria, CA) was used at a dilution of 1:300. The detection system was the Standard ABC kit (Vector Laboratories, Burlingame, CA), with 3,3'-diaminobenzidine (DAB) as the chromogen and a modified Harris hematoxylin counterstain.

To detect macrophage antigen 2 (MAC-2) expressing cells, slides were stream hydrated with citrate buffer for 20 minutes. The primary antibody, rat anti-Mac-2 (TIB-166, ATCC, Mannasas, VA) was used at a 1:200 dilution followed by a biotinylated goat-anti-rat IgG secondary antibody and developed with Streptavidin HRP (Biocare, Concord, CA).

Initially, survivor numbers were compared by the Mantel-Cox log-rank test. When statistical significance was found, pairwise analyses were performed using the Gehan-Breslow-Wilcoxon test. Differences in tissue virus titers and lung weights were statistically analyzed by the Tukey-Kramer multiple comparisons test. Lung hemorrhage scores were analyzed by the two-tailed Mann-Whitney U-test. Analyses were performed using the InStat® and Prism 5.0® computer software programs (GraphPad Software, San Diego, CA), comparing treated and placebo groups.

The magnitude of MERS-CoV viral load in nasopharyngeal secretions46 and blood47 has been directly correlated with higher mortality in some studies. The utility of upper respiratory samples is, however, not clear since MERS-CoV is a lower respiratory tract pathogen and the viral load in lower respiratory samples has minimal correlation to the risk of death.48

There remains a dearth of studies on the immunology of MERS-CoV infection, with even less information that compares cohorts of both MERS-CoV survivors and non-survivors, nor is there a significant literature regarding SARS-CoV immunology that may serve as a paradigm. For SARS-CoV, B cell immunity was shown to be short-lived with antibodies undetectable in up to 90% of survivors by 24 months49,50 whereas in contrast, T-cell responses were long-lived and persistent to at least 6 y.49 Importantly, mouse studies demonstrated that cytotoxic T-cell immunity against SARS-CoV was required for viral clearance and survival from lethal infection.51,52

The kinetics of the serologic response against MERS-CoV shows that binding and neutralizing antibodies appear at about day 10 of illness, reaching a peak a few days later.53 A small Saudi Arabian study of 7 MERS-CoV survivors demonstrated persistence of neutralizing antibodies for almost 3 y.54 The role of neutralizing antibodies in viral clearance is, however, not clear. A Korean study of 17 patients showed no clear difference in the pattern or timing of binding antibody development between those with severe vs. non-severe disease, whereas appearance of neutralizing antibodies was delayed by a few days in those with severe disease but once apparent, reached titers ≥ 1:320 more rapidly.53 Notably only 2 patients (1 with severe and 1 with non-severe disease) did not develop neutralizing antibodies greater than 1:20. A study of 37 persons from Saudi Arabia found that 24 of 27 (89%) of all patients with complete data demonstrated binding and neutralizing antibodies.48 Pairwise correlation found no association between the presence of neutralizing antibodies and viral clearance. Thus, the role for neutralizing antibodies in MERS-CoV disease outcomes is not established.

Finally, one could question whether subclinical or non-lethal infection provides long-term protective immunity against recurrent MERS-CoV infection. Considering the fact that camels have high sero-prevalence of MERS-CoV, it would be expected that camel workers would have recurrent MERS-CoV exposure. Yet, 2 large seroepidemiologic surveys of camel workers in Saudi Arabia found a low prevalence of anti-MERS-CoV antibodies55,56 suggesting that antibodies may in fact not be persistent. Moreover, the fact that many with camel exposure continue to present with MERS-CoV infection also suggests that prior exposures may not provide long-term immunity.

Viral-induced cytotoxicity was determined by measuring adenylate kinase activity in apical washes using a commercially available assay (Lonza, Inc.). Apical samples were centrifuged prior to freezing to remove any cellular contaminants present in the wash. Luminescence detected in samples from infected HAE were normalized to uninfected HAE and expressed as fold change over AK measured in uninfected (mock) HAE. Morphological assessment of cytotoxicity in HAE was performed with paraformaldehyde (PFA, 4%)-fixed histological sections (5 µm) stained with hematoxylin and eosin.

Design and conduct of an efficacy trial for MERS-CoV may be a daunting task as the epidemiology of MERS-CoV is vastly different from the start of the outbreak in 2012 - with fewer cases, that are scattered across Saudi Arabia. Except for a single large outbreak in Seoul Korea, there has been minimal transmission of MERS-CoV outside of Saudi Arabia.

While MERS-CoV remains endemic in Saudi Arabia with approximately 20–30 cases diagnosed monthly, vigilance in maintaining strict infection control procedures has significantly reduced new cases among healthcare workers (HCWs) and spread to patients in healthcare facilities. Nor have there been additional outbreaks outside of the Arabian Peninsula akin to the Korean epidemic of September 2015. Additionally, many incident infections occur in individuals without a clear epidemiologic link to a known case or to camels. All of these factors create challenges in the design of a definitive efficacy trial for any MERS-CoV vaccine.

Basic protocol designs include ring vaccination studies to prevent infection among direct contacts and studies to prevent incident infection groups at highest risk for MERS-CoV infection. Ring vaccination was successfully used in the Ebola epidemic,69 made possible by the fact that family and healthcare contacts were at high risk for infection. Transmission of MERS-CoV within family units has been documented,11 however, such cases appear to be more of an anomaly. In healthcare settings, infection control measures have significantly reduced spread between patients and to HCWs. Thus, a ring-vaccination strategy would require the enrollment of a large number of recruited families and contacts to reach a sufficient number of events to achieve statistical power.

A second study design is of population-based vaccination for those at highest risk for infection: HCWs, residents in towns and villages with the highest historical case rates, and those with camel contact. A key challenge is how to best identify those with past MERS-CoV exposure, and, of this group, to determine which individuals may have pre-existent protective B cell and/or T cell immunity. Whether any vaccine study should be restricted to non-immune individuals is an interesting question since it has already been demonstrated that a minority of individuals with repeated exposure to camels have detectable antibodies, suggesting that immunity may not be persistent.55,56 And the fact that camel exposure continues as a known risk for infection, further raises the question of whether non-lethal infection results in protective immunity and again, whether such immunity is persistent. Thus, studies in risk-groups could be stratified between those with or without documented MERS-CoV immune responses. Whether exposure should be defined by epidemiologic exposure or the presence of binding antibodies, neutralizing antibodies, or T cell responses is also unknown.

Finally, a third clinical trial design could focus on those at highest risk for severe infection. Such a study would more easily discern vaccine effectiveness since the primary outcome would compare morbidity and death between vaccine and placebo. However, those at highest risk for severe disease including the elderly and those with underlying illness such as cardiac, pulmonary, and renal disease,4,70 may limit vaccine immune responsiveness.

Any MERS-CoV vaccine has a key challenge as to the ability to conduct a definitive efficacy trial. The decrease in incident cases overall and the fact that primary cases are geographically separated are the 2 primary factors making such a trial difficult. An efficacy trial to prevent primary infection may be possible if restricted to on Saudi villages and towns with the greatest number of known cases. And since ongoing nosocomial spread is still documented, including a small outbreak in June 2017, a study to prevent infection in health care workers may be feasible. There is interest in a MERS-CoV camel vaccine that may both limit human disease and provide an alternative path to licensure via the animal rule, although vaccine development in camels presents its own unique challenges. As indicated by the epidemiology of infection, a MERS-CoV vaccine would primarily target the population in endemic countries, especially those in the health-care industry and those with contact with camels. Secondary markets exist for those traveling to (or from) the Arabian Peninsula, perhaps including those making pilgrimage to the Hajj and as a stockpile by governments against future outbreaks.

HAE maintained at either 32°C or 37°C for 72 hrs prior to sialic acid detection were washed, blocked with 3% BSA/PBS++ and probed with biotinylated SNA or MAA lectins to detect α2,6 and α2,3 SA, respectively (Vector Laboratories, Inc.; EY-Laboratories, Inc.; 1∶100). HAE were then fixed in 4% PFA and incubated with streptavidin-alexafluor 488 (Molecular Probes, Inc.; 1∶500) applied to the apical surface to detect lectin binding.

The marmoset study requires a sample size of 3 for adequate power to determine if the incidence of seroconversion is significantly greater than that which would be expected in the population. This sample size will allow the experimenter to detect seroconversion in at least 2 of 3 animals (66%) versus the expected population constant of <1% at a 95% confidence level using a one-tailed binomial test for proportions.

Viral kinetics were assessed using real-time RT-PCR data. Infection was defined as a challenge virus load of >106 copies/100 µL of nasal wash for at least 1 measurement, blocking/prevention was defined as a challenge virus load of <106 copies/100 µL of nasal wash for all measurements, and coinfection was defined as primary virus and challenge virus loads of >106 copies/100 µL of nasal wash for at least 1 day.

For statistical analysis, ferrets infected with the challenge virus were split into 2 groups: those in which primary infection persisted following challenge and those in which primary infection subsided and was below the threshold for all days following challenge. Control ferrets were excluded. The times from challenge to (1) the start of shedding of challenge virus and (2) the peak of shedding were calculated for each ferret, and the group median value was determined. The difference in median values between groups was analyzed using the Wilcoxon rank sum test, with the significance level set at an α of 0.05.

Linear regression was performed on the number of days from challenge to the start/peak of shedding for the challenge virus versus the number of days for which the primary infection persisted following challenge, and a 95% confidence interval (CI) for the gradient was obtained; this indicates the interval that the start/peak of shedding for the challenge virus was delayed for each day that the primary infection persisted following challenge. The null hypothesis, that there is no delay, was tested, with the significance level set at an α of 0.05.

The duration of infection with the challenge virus was compared for ferrets first infected and challenged with heterosubtypic influenza A viruses (when challenge was successful) and corresponding control ferrets; the difference in the median value was analyzed using the Wilcoxon rank sum test, with the significance level set at an α of 0.05. Statistical analysis was conducted using R, version 2.15.1.

To develop a non-human primate model of LACV infection for pathogenesis studies and for testing of future vaccine candidates, rhesus monkeys were inoculated with 105 PFU of biologically cloned (terminally diluted) human or mosquito LACV (LACV/human/1978-clone, LACV/mosquito/1978-clone). Illness was not observed following virus administration, and virus was not detected at any time in serum samples (Table 2). Virus was present at a low titer in lymph nodes on days 6, 8, and 12, however, virus replication in these tissues could not be identified by IHC staining. Despite the low level (or absence) of viremias and highly restricted replication in the tissues sampled, all monkeys developed neutralizing antibody responses that were first detected on days 6–8 indicating that the each monkey was infected. Twenty-eight days after inoculation, neutralizing antibody titers (PRNT60) for each group were in the range of 1:560 – 1:2186 (Table 2). Low-level cross-reactive antibodies were present in two monkeys (CL6E and DBOH) at the start of the experiment. A boost in antibody titer in these monkeys was not observed compared to other monkeys, suggesting that this experimental LACV infection was a primary infection.

Complete blood counts (CBC) with differential and blood chemistries were analyzed at each blood collection. Since LACV infection in monkeys was asymptomatic and also since differences between the four virus groups indicated in Table 2 were not observed, the CBC and blood chemistry data were averaged for the 16 animals to detect changes in blood values during the course of infection (Table 3). Days in which specific parameter values for a significant number of monkeys were greater than 1 standard deviation from normal appear boxed in Table 3 with mean values for each test shown. After day 6, the majority of monkeys experienced a slight anemia, which may in part be associated with the overnight fast in preparation for anesthesia prior to each blood collection. This analysis suggests that infection of major organs such as liver was minimal or absent.

To estimate the minimum dose required to infect a monkey, rhesus monkeys were inoculated with LACV/mosquito/1978-cl at 101 or 103 PFU subcutaneously. Blood was collected on days 0, 21, 28, and 42, and serum neutralizing antibody titers were determined. Mean neutralizing antibody titers were 1:355 and 1:82 for the 101 or 103 PFU dose groups, respectively, and all monkeys seroconverted by day 28 (PRNT60 ≥ 40) (Table 4). Thus, LACV is highly infectious for rhesus monkeys even at a dose of 101 PFU and results in the induction of a high level of neutralizing antibody. However, LACV infection did not result in identifiable clinical abnormalities in this group of 24 monkeys.

In this study we have demonstrated the susceptibility of marmosets to HTNV infection. Marmosets represent an attractive model for testing vaccines and therapeutics against HFRS-causing hantaviruses due to genetic similarities to humans and small size. Also, the model has a simple read-out of infection, i.e. robust antibody production as measured by N-ELISA and PsVNA, making the determination of protection by vaccine or passive transfer material, straightforward. Further optimization of the model, namely to determine the ID99, could prove important as a 1,000 PFU challenge dose could be excessively high and prohibit therapeutic effects of candidate medical countermeasures.

Overall the marmoset model is more similar to the ferret HFRS-causing hantavirus infection model than the hamster, though there are key differences. Like the ferret, no significant pathological abnormalities were noted, and no signs of renal failure were observed (S16 and S19 Figs). Serum chemistry values do not differ from the normal range with the exception of albumin, total bilirubin, and amylase. While these values fell outside the normal range, they did not change over the course of infection indicating the problem may lie in the reference values used. The Piccolo general chemistry panel used to evaluate the parameters is optimized for human testing, and therefore may be less than optimal for evaluating the marmoset, especially those parameters. Additionally, no infectious virus or viral genome was recovered from any organ at Day 30 post infection [79, 80]. This is not surprising, given the high levels of neutralizing antibodies present as early as 21 days post infection (Fig 8). Unlike the ferret, however, marmosets develop exceptionally high neutralizing antibody titers (10,240–20,480 by PRNT50 and 14,866–221,557 by PsVNA50), and display low-level serum viremia between two and four weeks post infection (Fig 8). The serum viremia is significantly lower than in hamsters infected with HTNV, where some animals displayed RT-PCR titers of >7log10, and in hamsters infected with ANDV, where infectious virus titers prior to death are > 6log10.

Despite not exhibiting clinical signs of disease, the model’s robust antibody response (as measured by PRNT, PsVNA and N-ELISA) make it a useful tool for evaluating vaccines and pre-or post-exposure therapeutics.

The logistical difficulties in pursuing standard vaccine evaluation have created significant interest in the possibility of controlled human infection models (CHIM). The conduct and planning for human challenge trials raises unique medical and ethical considerations. For Zika virus trials, one must balance the relative benign and self-limited infection experienced by the vast majority against the risk for less benign complications and transmission risks.

As reviewed above, published studies provide estimates that Zika virus infection is relatively asymptomatic and self-limited for the majority of individuals, however, methodology in these studies varies widely. In adults, two sets of complications warrant consideration for a proposed CHIM study. As reviewed above, a Guillain–Barre-like syndrome occurs in approximately 1 of 5000 Zika virus infections, whereas other neurologic complications such as meningoencephalitis, myelitis, and acute disseminated encephalomyelitis are rare. Second, and perhaps more important, is the risk of transmission to a sexual partner and the potential for infection during pregnancy. Zika virus commonly persists in the male urogenital tract for 3 months, and may persist in some individuals for up to 8 months. Some have considered limiting studies to non-pregnant females as Zika virus colonization of the female genital tract may be temporally limited. The fact that Zika virus is known to cause testicular atrophy in mice, raises yet another as yet theoretical concern for humans. These questions as well as the theoretical potential for vector-borne transmission were debated in detail in late 2016 with the conclusion that the benefits of a human challenge infection did not outweigh the risks, however, this analysis was performed just as the initial epidemic wave in the Americas was ending. The group published a follow-up in 2018. Despite the recognition that conducting a placebo-controlled vaccine trial had become significantly more difficult due to declining case rates, the group’s conclusion was essentially unchanged.

To address safety concerns, there has been work to develop attenuated viral strains deleted for potential neurotropic regions with a goal to prevent viremia. Whether the attenuated viral strain (rZIKV/D4Δ30-713) being tested in a Phase I study will serve as putative challenge strain is as yet undetermined.

Mice were anesthetized with ketamine/xylazine (50/5 mg/kg) by intraperitoneal (i.p.) injection for i.n. treatments and i.n. infection. I.p. infections required no anesthesia. Amounts of infecting virus (PFU/mouse) to achieve complete lethality varied with virus type, infection route, and infection volume, as follows: vaccinia (100-µl i.p.) - 5×106, vaccinia (50-µl i.n.) –1×105, cowpox (10-µl i.n.) –1×106, and cowpox (50-µl i.n.)- 5×105. A long-term (8-week) prophylaxis study that was performed used aged mice, which required an i.n. vaccinia virus challenge dose of 2.5×105 PFU/mouse to cause complete mortality. mDEF201 was administered i.n. to the nasal sinus or lung 24 h prior to vaccinia virus exposure for short-term prophylaxis, or 56 days prior to infection for long term prophylaxis. Placebo-treated mice were given saline by i.n. route. All animals were weighed every other day starting the day before and during the 21-day infection period. There were 10 mice in each mDEF201 or cidofovir treated group and 20 placebos per experiment.

In the long-term prophylaxis study, separate animals (5 mice per group) were maintained for determination of tissue virus titers, lung hemorrhage scores, and lung weights. These mice were sacrificed for removal of tissues after 5 days of infection. Lungs were given a hemorrhage score (color change from pink to plum which occurs regionally in the lung rather than gradually over the entire lung) ranging from 0 (normal) to 4 (entire lung affected). Lungs, spleens, livers, and snouts were weighed prior to homogenization that releases infectious virus for titration. Homogenization of soft tissues was performed in 1 ml of cell culture medium using a stomacher. Snouts were ground in 1 ml of medium using sterilized mortars and pestles. Samples were serially diluted in 10-fold increments and plaque titrated in 12-well microplates of Vero cells. Plaques were stained at three days with 0.2% crystal violet in 10% buffered formalin, followed by counting the plaques with the aid of a light box. Plaque numbers were converted to PFU per gram of infected tissue.

ELISA assay was used to quantify the levels of interferon-ɑ/β. Supernatant in each group was collected at 6, 12 and 24 h postinfection, and analyzed by ELISA kits (R & D Systems). According to previous description [37, 38], cells in positive control group were treated with poly I: C (Sigma) at the concentration of 10 μg/mL.

The production of rabbit polyclonal anti-NP antibody was described previously, and this antibody was used as a primary antibody for indirect immunofluorescence assay. A goat anti-rabbit IgG antibody conjugated to Alexa Fluor 488 or Alexa Fluor 568 was purchased from Invitrogen and used as a secondary antibody for indirect immunofluorescence assay. A polyclonal antibody against influenza A virus was obtained from 2-month-old female rabbit immunized with 250 µg of purified virions of influenza virus strain A/Puerto Rico/8/34. The generation of antibodies was boosted three times and used as neutralizing antibodies to block the influenza virus infection.

Ferrets were anaesthetized (12.5 mg/kg ketamine and 2.5 mg/kg Ilium Xylazil-20 in a 1:1 [v/v] mixture; Troy Laboratories) and inoculated by dropwise delivery of 103.5 TCID50 of virus in 0.5 mL into the nostrils. After infection, ferrets were housed individually in a high-efficiency particulate arrestance (HEPA)-filtered isolation unit. Blood samples were obtained from ferrets before the primary virus infection and at the termination of the experiment. Animals were weighed and visually inspected, and their temperature was measured daily by using implanted temperature transponders fitted to identification chips (LifeChip Bio-Thermo, Digivet). Nasal wash specimens were collected and stored as previously described. On the day of collection, RNA was extracted from 140 µL of nasal wash for real-time reverse transcription–polymerase chain reaction (RT-PCR) analysis.

Vaccine development against the Zika virus began in earnest in late 2015 following the reports of microcephaly in fetuses and infants in Brazil. Of note, the first demonstration of immunoprotection was as part of a 1953 study to define the ultrastructural characteristics of Zika virus, that found intramuscular vaccination of mice with infectious viral filtrates protected against cerebral infection.

Poland et al. provide a comprehensive listing of almost 40 Zika virus vaccines in development as of 2017. Diamond et al. discuss potential immunoreactive epitopes on the Zika envelope and provide further information on those vaccines that progressed into clinical trials. The large number of delivery platforms include live attenuated and inactivated whole-viruses; viral-vectored vaccines utilizing adeno-associated virus, vesicular stomatitis virus, measles virus, and dengue or Yellow Fever chimeric vaccines; DNA and mRNA vaccines; and peptide and protein subunit vaccines. Most have been assessed in animal models utilizing non-human primates and/or lethal challenge experiments involving immunosuppressed mice. As of 2017, six vaccines had advanced to Phase I studies. Since that time, two additional vaccines have entered into clinical trials (Table 1).

A total of three DNA vaccines have entered into human testing, including one that has advanced to Phase II (Table 1).

GLS-5700, a DNA vaccine encoding for the Zika virus prME genes designed as a consensus based on available Zika virus sequences through December 2015, was the first to enter into clinical trials. In pre-clinical studies, vaccinated mice and non-human primates were shown to develop B and T-cell immune responses against the Zika virus envelope and protected against development of neurologic disease and death in immunosuppressed, interferon α, β receptor deficient (IFNAR) mice. Moreover, histologic sections of brain tissue showed that vaccinated mice were without inflammatory infiltrates evident in non-immunized mice. Subsequent studies showed that the vaccine protected against testicular damage, testicular atrophy, spermatozoal damage, and infertility in mice. A Phase I study evaluated GLS-5700 administered via intradermal injection (ID), followed by electroporation (EP) at doses of either 1 or 2 mg per vaccinations followed immediately by electroporation at baseline, 4 weeks and 12 weeks. There were no serious adverse events (SAE) reported as part of the study, with the most frequent adverse events related to discomfort, pain, or swelling at the injection site. Seroconversion was observed in 83% of individuals after two vaccinations and 100% after three vaccinations with GLS-5700. Neutralizing antibodies were detected for 62% of participants in a Vero-cell (monkey kidney cell) assay, however, 95% of participants demonstrated the ability to neutralize infection of U87MG neuroblastoma cells. Vaccine responses were maintained through a year of follow-up. There was no difference in responses based on dose level. Notably, passive transfer of immune serum from vaccinated participants was able to protect 92% of IFNAR mice against lethal infection independent of the presence of Vero cell neutralizing antibodies. GLS-5700 is being evaluated as part of a second double-blind, placebo-controlled clinical trial (NCT02887482) performed in Puerto Rico. Analysis of the latter study is in progress.

Two additional DNA vaccines, based on the French Polynesian H/PF/2013 strain were developed as chimeras that included the JEV prM signal sequence followed by the Zika envelope (E) gene (VRC5283) or a similar construct with the terminal 98 amino acids of E, representing the stem and transmembrane regions, exchanged for the analogous JEV sequence (VRC5288). Both vaccines were immunogenic for mice and NHPs and protected >90% of NHPs against viremia at a dose of either 1 or 4 mg given twice. Both vaccine candidates were advanced into clinical trials with 4 mg of administered intramuscularly at weeks 0, 4 and 8 with vaccine VRC5288 administered by needle and syringe while VRC5283 was administered either as a single dose or split dose by needle and syringe or as a split dose given by the Pharmajet needle-free device. Seroconversion was 100% in the group administered vaccine with the needle-free device, less for vaccine administered as split-dose by needle and syringe, and lowest for vaccine given as a single injection with needle and syringe. The VRC5283 vaccine was advanced into Phase II studies in the Americas that utilized 140 clinical sites with clinical site selection guided by epidemiologic modeling. Long-term follow-up has not been reported.

Three inactivated vaccines have entered into clinical studies, of which clinical data for only ZPIV vaccine has been published. ZPIV is a whole inactivated virus vaccine of Puerto Rican strain PRVABC59. Studies in mice showed that a single vaccination given intramuscularly with alum generated antibody titers of approximately 3.7 log10 and fully protected Balb/C immunocompetent mice from viremia, whereas unvaccinated mice were unprotected and subcutaneously vaccinated mice were only partially protected against the Zika Brazil strain. A subsequent study in non-human primates vaccinated twice at four-week intervals with alum generated binding and microneutralization antibody titers of 3.54 and 3.55 log10, respectively, and complete protection against viremia and viruria following challenge with either Brazilian or Puerto Rican strains of Zika virus. ZPIV safety and immunogenicity was tested in three clinical trials to assess Zika vaccine responses relative to dose level, vaccination schedule, or following vaccination with either the Yellow Fever YF-VAX or Japanese encephalitis virus (JEV) IXARO vaccines to assess responses in a flavivirus-exposed population. There were no vaccine-associated SAEs reported. The most common adverse events were pain and tenderness at the injection site; no neurologic events were reported. Seroconversion was 92% using a cutoff for peak geometric mean titer of 1:10 and 77% using a titer of 1:100. Response rates after a single immunization were 11% and 5.5% using cutoffs of 1:10 or 1:100, respectively. Vaccine responses were observed through day 57. Passive transfer of purified IgG derived from 10 Zika immunized participants to groups of 5 Balb/C mice per participant provided sterilizing immunity against viremia for 82% (41 of 50) of mice overall, with viremia observed for one or more mice per group inoculated with sera from five (50%) individuals.

Clinical trial data for the other vaccines has not yet been reported as of the date of this monograph, however, pre-clinical data has been reported for three candidate vaccines. An mRNA vaccine that incorporates the prM-E genes of a Micronesian strain of Zika virus was created incorporating with or without four-point mutations in the fusion-loop segment of the DII region of the envelope gene that abolished binding of antibodies directed against the fusion-loop region to reduce the potential risk for antibody-dependent enhancement of infection. Immunization of AG129 mice with un-modified lipid nanoparticle-encapsulated vaccines mRNA was immunogenic and protective against lethal infection; immunization of C57BL/6 immunocompetent mice followed by treatment with anti-ifnar1 blocking antibody showed protection against viremia in approximately 60% of animals. PIZV, an inactivated vaccine derived from Puerto Rican strain PRVABC59 selected as without passage-related mutations, protected against lethal Zika virus challenge in AG129 mice when administered with alum adjuvant. A measles-vectored vaccine encoding the Zika virus prME was shown to lessen viremia in pregnant IFNAR mice and prevented clinical disease in mouse pups. Preclinical data for VLA-1601 inactivated viral vaccines and the rZIKV/D4Δ30-713 live virus vaccine has not yet been reported.

Immunofluorescence and immunohistochemistry staining was done on ud-NHBE and wd-NHBE cells. The transwell cultures were collected and fixed with 10% formalin, paraffin embedded, and then cross-sectioned for histological examination. Slides were stained using hematoxylin-eosin. Ciliated cells were further identified by FITC-conjugated β-tubulin antibody (Sigma, Saint Louis, USA) and goblet cells were identified by biotinylated MUC5AC antibody (Invitrogen, San Francisco, USA). Moreover, the expression of human airway trypsin-like (HAT) protease was detected by RT-qPCR using the mRNA extracted from the NHBE cultures. Human bronchial tissue was also stained with β-tubulin and MUC5AC. Tissues were obtained from lobectomy or pneumonectomy specimens of patients having surgical resection of lung tissue. The study was approved by The Hong Kong University and Hospital Authority (Hong Kong West) Institutional Review Board.

Data are presented as mean ± standard deviation (SD), where applicable. Inferential statistical analysis was performed by Student’s t-test, Welch’s t-test, and One-Way ANOVA followed by Tukey’s test. p values < 0.05 were considered statistically significant.

Cells in each group used for western blot analysis were collected at 12, 24 36 h after treatment and lysed with RIPA lysis buffer (Cell Signaling Technology). The cell lysates were centrifuged and the resultant supernatants were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Roche). The membranes were blocked with 5% skim milk and probed with a monoclonal antibody to β-actin (Santa Cruz Biotechnology), IFITM1 (Sigma-Aldrich). After a further incubation with peroxidase (HRP) conjugated secondary antibody (ORIGENE). Proteins were visualized using enhanced chemiluminescence. The relative protein level of IFITM1 to β-actin was analyzed by Image J software.

Lung cell suspensions were incubated with anti-Fc block (anti-mouse CD16/CD32) to reduce non-specific antibody binding for 10 min. prior to staining for 1 hr with fluorophore-conjugated antibodies (BD Biosciences, San Diego, CA) specific for immune cell populations according to standard protocols and included: CD11b-PE (pan-macrophage), CD11c-APC (pan-dendritic cell), Ly6G/C-FITC (neutrophil), CD4-PE and CD8-APC T cell markers (Table 2, Figure 2). Cells were washed twice with PBS and fixed overnight at 4°C with 2% paraformaldehyde. Samples were safety tested for infectious virus and removed from the BSL3+ laboratory. Flow cytometry was performed on a FACSAria flow cytometer (BD Biosciences). To further characterize primary mouse and human macrophage and dendritic cells we utilized the following fluorescently conjugated antibodies for flow cytometric analysis: MHC II (I-A/I-E)-PE, HLA-DR-PE, CD14-FITC, CD40-FITC, CD80-FITC, CD83-APC, and CD86-APC (BD Biosciences) (Figures 4 and 6).

Animal tissues were fixed in 10% phosphate-buffered formalin for pathologic examination. They were then processed for paraffin embedding and cut into 3-µm-thick serial sections. One section from each tissue sample was subjected to standard hematoxylin-and-eosin staining, while another was processed for immunohistochemistry with a mouse monoclonal antibody for type A influenza virus NP antigen that reacts comparably with all of the test viruses. This antibody was produced in our laboratory and used for immunohistochemistry at 1:1000 dilution. Specific antigen-antibody reactions were visualized by use of 3,3′-diaminobenzidine tetrahydrochloride staining and a Dako EnVision system (Dako Co. Ltd., Tokyo, Japan).

Two- to three-year-old male cynomolgus macaques, weighing 2.0–3.0 kg and serologically negative by neutralization assays for currently circulating human influenza viruses, were obtained from Cambodia (obtained from Japan Laboratory Animals, Inc., Tokyo, Japan). All experiments with macaques were performed in accordance with the Regulation on Animal Experimentation Guidelines at Kyoto University and were approved by the Committee for Experimental Use of Nonhuman Primates in the Institute for Virus Research, Kyoto University.

For the aerosol inoculation (aerosol method group), animals were anesthetized with ketamine via intramuscular injection and, by using the ultrasonic nebulizer NE-U17 (Omron Healthcare Co, Ltd, Japan), 4 ml of 107 PFU/ml of virus was aerosolized and administered to each animal via an inhalation mask, which is not a tight-fitting mask and allows the individual to breathe the aerosol mist through the nose and mouth spontaneously. For virus inoculation through multiple routes, that is, the conventional method (the conventional method group), animals were anesthetized with ketamine via intramuscular injection and inoculated with a suspension containing a total of 4 × 107 PFU of virus through a combination of the intratracheal (2.2 ml), intranasal (0.4 ml per nostril), ocular (0.1 ml per eye) and oral (0.4 ml each to the left and right tonsil) routes. Body temperature was monitored at 0, 1, 3, and 4 days post-infection by use of a rectal thermometer.

At the indicated timepoints post-infection, two or four macaques per group were euthanized for virologic and pathologic examinations. The virus titers in various organs and nasal swabs were determined by using plaque assays in MDCK cells.

Mouse splenocytes were stained with antibodies to B220, GL-7, CD11c, CD11b, F4/80 and ICAM-1 (all BioLegend), Fas (BD biosciences), and FDC-M2 (ImmunoKontact). Fixable Viability Stain 510 (BD biosciences) was used for all flow cytometry experiments. Mouse lung cells were stained with antibodies to CD11c, CD11b and F4/80 (all BioLegend). Splenocytes and lung cells were fixed using the Transcription Factor Buffer Set (BD biosciences) according to the manufacturer’s protocol. All samples were run on an LSR Fortessa (BD biosciences) and analyzed using FlowJo (Tree Star).

Cells were seeded into 12-well plates for 24 h and then treated with various concentrations of human IFN-β1a or mouse IFN-β (PBL) as indicated in each experiment. Cells were infected with Candid#1 JUNV at an MOI of 3 PFU/cell. IFNs were supplemented after virus infection. Protein lysates were prepared in 2x Laemmli sample buffer at 1 and 2 days p.i. from MEF cells and A549 cells, or from Vero cells at 2 days p.i.. Protein samples were resolved on 4–20% SDS-PAGE gel and transferred to PVDF membranes using Mini Trans-Blot Electrophoretic Transfer Cell apparatus (Bio-Rad, CA). Membranes were incubated with primary antibodies overnight at 4°C and then with appropriate secondary antibodies for 1 h at room temperature. Proteins were visualized with ECL Western Blotting Detection Reagents (GE, NJ) according to the manufacturer's instruction. Viral NP protein was detected with a monoclonal mouse anti-JUNV NP antibody (AG12, BEI). Equal loading of samples was confirmed by immunoblotting of the same membranes with an antibody to β-actin (sc-1616, Santa Cruz). Secondary antibodies HRP-conjugated Goat anti-mouse IgG (115-035-146, Jackson Immunology) and HRP-conjugated donkey anti-goat IgG (sc-2020, Santa Cruz) were used.

The lectin Sambucus nigra agglutinin (SNA) used primarily detects Sia-α2-6-linkages and Maackia amurensis lectin (MAL)-I and MAL-II which identifies two Sia-α2-3Gal linkages: Sia-α2-3Galβ1-4GlcNAc and Sia-α2-3Galβ1-3GalNAc respectively. The antigen retrieval and staining of the paraffin sections of the NHBE cells and human bronchial tissues were performed as described,. Briefly, the sections were microwaved in citrate buffer and blocked with H2O2 in Tris buffered saline and with avidin/biotin blocking kit (Vector, Burlingame). They were then incubated with horseradish peroxidase conjugated SNA (EY Laboratories), biotinylated MAL-I (Vector, Burlingame) and 1∶100 biotinylated MAL-II (Vector, Burlingame) and blocked with 1% bovine serum albumin and then incubated with strep-ABC complex (Dako Cytomation, Cambridgeshire, UK). Development was performed using the AEC substrate kit (Vector, Burlingame), the nuclei were counterstained with Mayer's hematoxylin and then the sections were dried and mounted with DAKO aqueous mount (Dako Cytomation, Cambridgeshire, UK).