A Recombinant Subunit Based Zika Virus Vaccine Is Efficacious in Non-human Primates

Zika Virus (ZIKV), a virus with no severe clinical symptoms or sequelae previously associated with human infection, became a public health threat following an epidemic in French Polynesia 2013–2014 that resulted in neurological complications associated with infection. Although no treatment currently exists, several vaccines using different platforms are in clinical development. These include nucleic acid vaccines based on the prM-E protein from the virus and purified formalin-inactivated ZIKV vaccines (ZPIV) which are in Phase 1/2 clinical trials. Using a recombinant subunit platform consisting of antigens produced in Drosophila melanogaster S2 cells, we have previously shown seroconversion and protection against viremia in an immunocompetent mouse model. Here we demonstrate the efficacy of our recombinant subunits in a non-human primate (NHP) viremia model. High neutralizing antibody titers were seen in all protected macaques and passive transfer demonstrated that plasma from these NHPs was sufficient to protect against viremia in mice subsequently infected with ZIKV. Taken together our data demonstrate the immunogenicity and protective efficacy of the recombinant subunit vaccine candidate in NHPs as well as highlight the importance of neutralizing antibodies in protection against ZIKV infection and their potential implication as a correlate of protection

Development, optimization and validation of microsphere based luminex assays for identification of West Nile virus and dengue virus infections

M.S. University of Hawaii at Manoa 2013.Includes bibliographical references.Purpose: West Nile virus (WNV) and dengue virus (DENV) are emerging arthropod-borne flaviviruses that represent an immense global health problem. Therefore, there is need for improved diagnosis of these infections for timely patient management, to prevent the spread of flaviviral infections as well as to understand the virus and antibody dynamics in humans as well as animal models. In this study, Luminex-based WNV E-protein microsphere immunoassay (MIA) was developed and optimized for detection of anti-WNV IgG and IgM antibodies in mice using assay parameters such as serum heat-inactivation (HI) and-dilution. In addition, an in-house newly developed Luminex-based assay for detection of anti-DENV IgG and IgM antibodies was further validated using different serum panels and was further optimized for improved sensitivity and specificity which is critical for accurate diagnosis of DENV infection. Similarly, inhouse newly developed Polymerase Chain Reaction-Microsphere Bead Assay (PCR-MBA) was further validated for detection of DENV serotypes and WNV. Methods: Magnetic carboxylated microspheres were coupled to purified rWNV-E protein and WNV E-MIA was conducted using serial dilutions of HI and non-HI (NHI) serum samples collected at days 0, 3, 6, 8, 10 and 24 from mice inoculated with WNV. In-house newly developed DENV MIA was validated using different serum panels from Hawaii, Vietnam and Niue as compared to gold standard PRNT assay and in-house DENV IgM Capture ELISA (MAC-ELISA) and a U.S. FDA approved InBios DENV IgM Capture ELISA. To improve the specificity and sensitivity of DENV MIA, the effect of assay parameters such as serum dilution, the use of alternative blocking agents such as PVA, PVP and 5-10% animal serum in serum diluents as well as assay buffers and the type and concentration of secondary antibody on the non-specific binding was studied. An in-house newly developed PCR-MBA was further validated for differential detection of DENV serotypes using CDC DENV panel samples as compared to gold standard CDC DENV 1-4 real-time RT-PCR assay. For validation of PCR-MBA for detection of WNV, WNV cDNA was diluted serially (10-fold dilutions) from 3 × 107 PFU/ml to 0.3 PFU/ml and WNV specific qRT-PCR and PCR-MBA was conducted. Data was analyzed to assess the sensitivity and specificity of PCRMBA for detection of WNV as well as for differential detection of DENV serotypes. Results: In WNV E-MIA, serum HI significantly enhanced detection of IgM and IgG antibodies as compared to NHI serum. Anti-WNV IgM and IgG antibodies in HI sera were detected earlier at day 3 and IgM antibodies persisted up to day 24 after infection. HI serum at 1:20 dilution was found to be optimal for detection of both IgM and IgG antibodies as compared to higher serum dilutions. Further, addition of exogenous complement to the HI serum decreased the WNV E-MIA sensitivity. Results of validation of DENV MIA demonstrated that DENV MIA is 100% and 71-100% sensitive for detection of anti-DENV IgG and IgM antibodies, respectively from human serum samples. However, the specificity of DENV MIA was found to be 33-100% and 11-98% for detection of anti-DENV IgG and IgM antibodies respectively. The low specificity of DENV MIA for samples from Vietnam was not due to syphilis antibodies present in these serum samples. Use of alternative blocking agents such as PVA and PVP, 5-10% animal serum in serum diluents did not increase the specificity of DENV IgM MIA. Higher serum dilution as well as pretreatment of serum samples with high concentration of BSA or BSA-coated beads reduced the assay interference to some extent in DENV IgM MIA. The use of monoclonal IgM secondary antibody for detection of anti-DENV IgM antibodies resulted in low BSA IgM MFI for IgM false-positive samples as compared to polyclonal IgM secondary antibody in DENV MIA. However, the IgM MFI for true IgM positive samples was reduced using monoclonal antibody as compared to polyclonal secondary antibody. The specificity of PCR-MBA for detection of four DENV serotypes as compared to 'gold standard' CDC DENV 1-4 real time RT-PCR assay was found to be 100%, whereas the sensitivity of PCR-MBA was found to be varied from 50-100% for DENV-1, 29-100% for DENV-2, 100% for DENV-3 and 80-100% for DENV-4 using CDC DENV panel samples. PCR-MBA for detection of WNV is specific and more sensitive as compared to WNV-specific qRT-PCR. Moreover, low background MFI was observed for the other arboviruses included in PCR-MBA and thus indicated negligible probe cross reactivity for the DENV serotypes and WNV. Conclusions: Serum-HI and optimal dilution enhanced WNV E-MIA sensitivity by eliminating the complement interference. This optimized WNV E-MIA can be used for detecting low-titer anti-WNV antibodies during early and late phase of infection in mice. Validation of DENV MIA using different serum panels demonstrated that sensitivity and specificity of immunoassays may differ according to the origin of samples. Minimizing the serum concentration as well as the absorption of sera with high concentration of BSA or BSA coated beads decreased the non-specific binding in DENV IgM MIA. Moreover, the results of this study conclude that sample pretreatment or the use of alternative blocking agents will vary for immunoassays. Therefore, it is important to optimize the blocking conditions for newly developed immunoassays to avoid the falsepositive results. PCR-MBA efficiently detected and differentiated the DENV serotypes from CDC DENV panel samples as well as WNV. PCR-MBA can be further optimized for improved sensitivity for detection of dengue serotypes and WNV by changing the concentration of probe coupled, concentration of the input RNA, hybridization temperature and time

Infection with Non-Lethal West Nile Virus Eg101 Strain Induces Immunity that Protects Mice against the Lethal West Nile Virus NY99 Strain

Herein we demonstrate that infection of mice with West Nile virus (WNV) Eg101 provides protective immunity against lethal challenge with WNV NY99. Our data demonstrated that WNV Eg101 is largely non-virulent in adult mice when compared to WNV NY99. By day 6 after infection, WNV-specific IgM and IgG antibodies, and neutralizing antibodies were detected in the serum of all WNV Eg101 infected mice. Plaque reduction neutralization test data demonstrated that serum from WNV Eg101 infected mice neutralized WNV Eg101 and WNV NY99 strains with similar efficiency. Three weeks after infection, WNV Eg101 immunized mice were challenged subcutaneously or intracranially with lethal dose of WNV NY99 and observed for additional three weeks. All the challenged mice were protected against disease and no morbidity and mortality was observed in any mice. In conclusion, our data for the first time demonstrate that infection of mice with WNV Eg101 induced high titers of WNV specific IgM and IgG antibodies, and cross-reactive neutralizing antibodies, and the resulting immunity protected all immunized animals from both subcutaneous and intracranial challenge with WNV NY99. These observations suggest that WNV Eg101 may be a suitable strain for the development of a vaccine in humans against virulent strains of WNV

Effect of Serum Heat-Inactivation and Dilution on Detection of Anti-WNV Antibodies in Mice by West Nile Virus E-protein Microsphere Immunoassay

<div><p>Immunopathogenesis studies employing West Nile virus (WNV) mice model are important for the development of antivirals and vaccines against WNV. Since antibodies produced in mice early during WNV infection are essential for clearing virus from the periphery, it is important to detect early and persistent anti-WNV antibodies. ELISA and plaque reduction neutralization tests are traditionally used for detection of anti-WNV antibodies and WNV-neutralizing antibodies, respectively. Although these assays are sensitive and specific, they are expensive and time consuming. Microsphere immunoassays (MIA) are sensitive, specific, allow for high throughput, are cost effective, require less time to perform than other methods, and require low serum volumes. Several assay parameters such as serum heat-inactivation (HI) and dilution can alter WNV MIA sensitivity. We examined the effect of these parameters on WNV E-protein MIA (WNV E-MIA) for the enhanced detection of anti-WNV IgM and IgG antibodies. WNV E-MIA was conducted using serial dilutions of HI and non-HI (NHI) serum collected at various time points from mice inoculated with WNV. HI significantly enhanced detection of IgM and IgG antibodies as compared to NHI serum. WNV IgM and IgG antibodies in HI sera were detected earlier at day 3 and IgM antibodies persisted up to day 24 after infection. HI serum at 1∶20 dilution was found to be optimal for detection of both IgM and IgG antibodies as compared to higher-serum dilutions. Further, addition of exogenous complement to the HI serum decreased the WNV E-MIA sensitivity. These results suggest that serum-HI and optimal dilution enhance WNV E-MIA sensitivity by eliminating the complement interference, thereby detecting low-titer anti-WNV antibodies during early and late phases of infection. This improved MIA can also be readily employed for detection of low-titer antibodies for detection of other infectious agents and host proteins.</p> </div

Effect of serum heat-inactivation on the detection of anti-WNV IgM and IgG antibodies in mice.

<p>Adult C57BL/6 mice were inoculated subcutaneously with 100 PFU of WNV. Mice (n = 10 per time point) were bled at 0, 3, 6, 8, 10 and 24 days after infection and serum was separated, and same time point serum was pooled. Serum samples were HI at 56°C for 30 min. NHI and HI sera were diluted 1∶20 in PBS-1% BSA and were tested by WNV E-MIA for the presence of anti-WNV (<b>A</b>) IgM and (<b>B</b>) IgG antibodies. Results are reported as MFI per 100 microspheres. Data are expressed as MFI ± SD and is representative of three independent experiments conducted in duplicate. Dotted line indicates the cutoff value. HI serum is depicted by red line and NHI serum by blue line.</p

Effect of serum dilution on the detection of anti-WNV IgM and IgG antibodies in mice.

<p>NHI and HI sera at indicated time-points after infection were diluted serially from 1∶20 to 1∶160 in PBS-1% BSA and were tested by WNV E-MIA for the presence of anti-WNV (<b>A and B</b>) IgM and (<b>C and D</b>) IgG antibodies. Data are expressed as MFI ± SD and is representative of two independent experiments conducted in duplicate. Dotted line indicates the cutoff value. HI serum is depicted by red line and NHI serum by blue line.</p

Effect of exogenous addition of complement to heat-inactivated mice serum in WNV E-MIA.

<p>HI serum at indicated time-points after infection was diluted 1∶20 in PBS-1% BSA and 4 U of reconstituted guinea pig complement (C’) was added to 240 µL-diluted serum. Also, HI serum after addition of C’ was again heat-inactivated at 56°C for 30 min to inactivate the complement. HI serum without C’, with C’ and heat inactivation of HI serum with C’ were tested by WNV E-MIA for detection of anti-WNV (<b>A</b>) IgM and (<b>B</b>) IgG antibodies. Data are expressed as MFI ± SD and is representative of two independent experiments conducted in duplicate. Dotted line indicates the cutoff value. HI serum is depicted by red line.</p

Impaired virus clearance, compromised immune response and increased mortality in type 2 diabetic mice infected with West Nile virus.

Clinicoepidemiological data suggest that type 2 diabetes is associated with increased risk of West Nile virus encephalitis (WNVE). However, no experimental studies have elucidated the role of diabetes in WNV neuropathogenesis. Herein, we employed the db/db mouse model to understand WNV immunopathogenesis in diabetics. Nine-week old C57BL/6 WT and db/db mice were inoculated with WNV and mortality, virus burden in the periphery and brain, and antiviral defense responses were analyzed. db/db mice were highly susceptible to WNV disease, exhibited increased tissue tropism and mortality than the wild-type mice, and were unable to clear the infection. Increased and sustained WNV replication was observed in the serum, peripheral tissues and brain of db/db mice, and heightened virus replication in the periphery was correlated with enhanced neuroinvasion and replication of WNV in the brain. WNV infection in db/db mice was associated with enhanced inflammatory response and compromised antiviral immune response characterized by delayed induction of IFN-α, and significantly reduced concentrations of WNV-specific IgM and IgG antibodies. The compromised immune response in db/db mice correlated with increased viremia. These data suggest that delayed immune response coupled with failure to clear the virus leads to increased mortality in db/db mice. In conclusion, this study provides unique mechanistic insight into the immunopathogenesis of WNVE observed in diabetics and can be used to develop therapeutics for the management of WNVE among diabetic patients

Body weight and intraperitoneal glucose tolerance test in WT and <i>db/db</i> mice.

<p>(A) Body weight in grams of nine-week old WT and <i>db/db</i> mice. (B) Blood glucose levels in the WT and <i>db/db</i> mice during IGTT. The data expressed are the mean concentration (mg/dL) ± SEM of the blood glucose levels and are representative of two independent experiments. *p<0.0001.</p