Background Autochthonous transmission of Zika virus (ZIKV) has been reported in 87 countries since 2015. Although most infections are mild, there is risk of Guillain-Barré syndrome and adverse pregnancy outcomes. Vaccines are urgently needed to prevent Zika, but sufficient understanding of humoral responses and tools to assess ZIKV-specific immunity are lacking. Methods We developed a blockade-of-binding (BOB) ELISA using A9E and G9E, two strongly neutralising ZIKV-specific monoclonal antibodies, which do not react with dengue virus. Receiver operating characteristic curve analysis assessed A9E and G9E BOB serodiagnostic performance. BOB was then applied to samples from a surveillance cohort in Risaralda, Colombia, and phase 1 ZIKV vaccine trial samples, comparing results against traditional serologic tests. Findings In the validation sample set (n = 120), A9E BOB has a sensitivity of 93.5% (95% CI: 79.3, 98.9) and specificity 97.8 (95% CI: 92.2, 99.6). G9E BOB had a sensitivity of 100% (95% CI: 89.0, 100.0) and specificity 100% (95% CI: 95.9, 100). Serum from natural infections consistently tested positive in these assays for up to one year, and reactivity tracks well with ZIKV infection status among sera from endemic areas with complicated flavivirus exposures. Interestingly, a leading ZIKV vaccine candidate elicited minimal BOB reactivity despite generating neutralising antibody responses. Interpretation In conclusion, A9E and G9E BOB assays are sensitive and specific assays for detecting antibodies elicited by recent or remote ZIKV infections. Given the additional ability of these BOB assays to detect immune responses that target different epitopes, further development of these assays is well justified for applications including flavivirus surveillance, translational vaccinology research and as potential serologic correlates of protective immunity against Zika.

Standard molecular detection of many pathogens, in particular RNA viruses, requires appropriate handling in the field for preserving the quality of the sample until processing. This could be challenging in remote tropical areas. Dengue virus (DENV), chikungunya virus (CHIKV), and Zika virus (ZIKV) are RNA viruses, prominent among the causes of fever in the tropics. We aimed to test the stability of arboviral RNA in contrived dried blood spots prepared on Whatman 903 Protein saver cards as a means of sample collection and storage. We were able to detect DENV, CHIKV, and ZIKV by real-time RT-PCR up to 180 days after card inoculation with stable Ct values across the study period. Our study supports dried blood spots (DBS) on protein saver cards as a platform for stable detection of arboviral RNA of sufficient quality to be used in diagnostic RT-PCR assays and next generation sequencing.

Background: Western (WEEV), eastern (EEEV), and Venezuelan (VEEV) equine encephalitis viruses are mosquito-borne pathogens classified as potential biological warfare agents for which there are currently no approved human vaccines or therapies. We aimed to evaluate the safety, tolerability, and immunogenicity of an investigational trivalent virus-like particle (VLP) vaccine, western, eastern, and Venezuelan equine encephalitis (WEVEE) VLP, composed of WEEV, EEEV, and VEEV VLPs. Methods: The WEVEE VLP vaccine was evaluated in a phase 1, randomised, open-label, dose-escalation trial at the Hope Clinic of the Emory Vaccine Center at Emory University, Atlanta, GA, USA. Eligible participants were healthy adults aged 18–50 years with no previous vaccination history with an investigational alphavirus vaccine. Participants were assigned to a dose group of 6 μg, 30 μg, or 60 μg vaccine product and were randomly assigned (1:1) to receive the WEVEE VLP vaccine with or without aluminium hydroxide suspension (alum) adjuvant by intramuscular injection at study day 0 and at week 8. The primary outcomes were the safety and tolerability of the vaccine (assessed in all participants who received at least one administration of study product) and the secondary outcome was immune response measured as neutralising titres by plaque reduction neutralisation test (PRNT) 4 weeks after the second vaccination. This trial is registered at ClinicalTrials.gov, NCT03879603. Findings: Between April 2, 2019, and June 13, 2019, 30 trial participants were enrolled (mean age 32 years, range 21–48; 16 [53%] female participants and 14 [47%] male participants). Six groups of five participants each received 6 μg, 30 μg, or 60 μg vaccine doses with or without adjuvant, and all 30 participants completed study follow-up. Vaccinations were safe and well tolerated. The most frequently reported symptoms were mild injection-site pain and tenderness (22 [73%] of 30) and malaise (15 [50%] of 30). Dose-dependent differences in the frequency of pain and tenderness were found between the 6 μg, 30 μg, and 60 μg groups (p=0·0217). No significant differences were observed between dosing groups for any other reactogenicity symptom. Two adverse events (mild elevated blood pressure and moderate asymptomatic neutropenia) were assessed as possibly related to the study product in one trial participant (60 μg dose with alum); both resolved without clinical sequelae. 4 weeks after second vaccine administration, neutralising antibodies were induced in all study groups with the highest response seen against all three vaccine antigens in the 30 μg plus alum group (PRNT80 geometric mean titre for EEEV 60·8, 95% CI 29·9–124·0; for VEEV 111·5, 49·8–249·8; and for WEEV 187·9, 90·0–392·2). Finally, 4 weeks after second vaccine administration, for all doses, the majority of trial participants developed an immune response to all three vaccine components (24 [83%] of 29 for EEEV; 26 [90%] of 29 for VEEV; 27 [93%] of 29 for WEEV; and 22 [76%] of 29 for EEEV, VEEV, and WEEV combined). Interpretation: The favourable safety profile and neutralising antibody responses, along with pressing public health need, support further evaluation of the WEVEE VLP vaccine in advanced-phase clinical trials.

The recent Zika virus (ZIKV) epidemic in the Americas has revealed rare but serious manifestations of infection. ZIKV has emerged in regions endemic for dengue virus (DENV), a closely related mosquito-borne flavivirus. Cross-reactive antibodies confound studies of ZIKV epidemiology and pathogenesis. The immune responses to ZIKV may be different in people, depending on their DENV immune status. Here, we focus on the human B cell and antibody response to ZIKV as a primary flavivirus infection to define the properties of neutralizing and protective antibodies generated in the absence of preexisting immunity to DENV. The plasma antibody and memory B cell response is highly ZIKV type-specific, and ZIKV-neutralizing antibodies mainly target quaternary structure epitopes on the viral envelope. To map viral epitopes targeted by protective antibodies, we isolated 2 type-specific monoclonal antibodies (mAbs) from a ZIKV case. Both mAbs were strongly neutralizing in vitro and protective in vivo. The mAbs recognize distinct epitopes centered on domains I and II of the envelope protein. We also demonstrate that the epitopes of these mAbs define antigenic regions commonly targeted by plasma antibodies in individuals from endemic and nonendemic regions who have recovered from ZIKV infections.

Background Current urban vector control strategies have failed to contain dengue epidemics and to prevent the global expansion of Aedes-borne viruses (ABVs: dengue, chikungunya, Zika). Part of the challenge in sustaining effective ABV control emerges from the paucity of evidence regarding the epidemiological impact of any Aedes control method. A strategy for which there is limited epidemiological evidence is targeted indoor residual spraying (TIRS). TIRS is a modification of classic malaria indoor residual spraying that accounts for Aedes aegypti resting behavior by applying residual insecticides on exposed lower sections of walls (< 1.5 m), under furniture, and on dark surfaces. Methods/design We are pursuing a two-arm, parallel, unblinded, cluster randomized controlled trial to quantify the overall efficacy of TIRS in reducing the burden of laboratory-confirmed ABV clinical disease (primary endpoint). The trial will be conducted in the city of Merida, Yucatan State, Mexico (population ~ 1million), where we will prospectively follow 4600 children aged 2–15 years at enrollment, distributed in 50 clusters of 5 × 5 city blocks each. Clusters will be randomly allocated (n = 25 per arm) using covariate-constrained randomization. A “fried egg” design will be followed, in which all blocks of the 5 × 5 cluster receive the intervention, but all sampling to evaluate the epidemiological and entomological endpoints will occur in the “yolk,” the center 3 × 3 city blocks of each cluster. TIRS will be implemented as a preventive application (~ 1–2 months prior to the beginning of the ABV season). Active monitoring for symptomatic ABV illness will occur through weekly household visits and enhanced surveillance. Annual sero-surveys will be performed after each transmission season and entomological evaluations of Ae. aegypti indoor abundance and ABV infection rates monthly during the period of active surveillance. Epidemiological and entomological evaluation will continue for up to three transmission seasons. Discussion The findings from this study will provide robust epidemiological evidence of the efficacy of TIRS in reducing ABV illness and infection. If efficacious, TIRS could drive a paradigm shift in Aedes control by considering Ae. aegypti behavior to guide residual insecticide applications and changing deployment to preemptive control (rather than in response to symptomatic cases), two major enhancements to existing practice. Trial registration ClinicalTrials.gov NCT04343521. Registered on 13 April 2020. The protocol also complies with the WHO International Clinical Trials Registry Platform (ICTRP) (Additional file 1).

Background Zika virus caused thousands of congenital anomalies during a recent epidemic. Because Zika emerged in areas endemic for dengue and these related flaviviruses elicit cross-reactive antibodies, it is challenging to serologically monitor pregnant women for Zika infection. Methods A prospective cohort of 253 pregnant women was established in León, Nicaragua. Women were followed during prenatal care through delivery. Serologic specimens were obtained at each visit, and birth outcome was recorded. Established flavivirus serologic methods were adapted to determine Zika seroprevalence, and a stepwise testing algorithm estimated timing of Zika infection in relation to pregnancy. Results Zika seroprevalence was approximately 59% among women tested. Neutralization testing was highly concordant with Zika NS1 BOB results. Per study algorithm, 21% (40/187) of women were classified as experiencing Incident ZIKV infection during pregnancy. Importantly, the Incident ZIKV group included mostly women pregnant during the 2016 Zika epidemic peak and the only 3 subjects in the cohort with RT-PCR-confirmed infections. Approximately 17% of births had complications; 1.5% (3/194) manifesting clinical criteria of congenital Zika syndrome, one was RT-PCR-confirmed as a case of congenital Zika syndrome. Adverse birth outcome did not correlate with timing of Zika infection. Conclusions By leveraging prenatal care systems, we developed a simple algorithm for identifying women who were likely infected by Zika during pregnancy.

Little is known about enduring memory B cell (MBC) responses to Zika virus (ZIKV) and their relationship with circulating antibodies. Here we comprehensively assess MBC frequency and specificity alongside serum binding and neutralizing antibody responses to ZIKV ~2 weeks and ~8 months postinfection in 31 pediatric subjects with 0, 1 or >1 prior infections with the related dengue virus (DENV). ZIKV infection elicits a robust type-specific MBC response, and the majority of late convalescent anti-ZIKV serum neutralizing activity is attributable to ZIKV-specific antibodies. The number of prior DENV infections does not influence type-specific or cross-reactive MBC responses, although ZIKV has the highest cross-reactivity with DENV3. DENV cross-reactive MBCs expanded by ZIKV infection decline in number and proportion by late convalescence. Finally, ZIKV induces greater cross-reactivity in the MBC pool than in serum antibodies. Our data suggest immunity to DENV only modestly shapes breadth and magnitude of enduring ZIKV antibody responses.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that first emerged in late 2019 is responsible for a pandemic of severe respiratory illness. People infected with this highly contagious virus can present with clinically inapparent, mild, or severe disease. Currently, the virus infection in individuals and at the population level is being monitored by polymerase chain reaction (PCR) testing of symptomatic patients for the presence of viral RNA. There is an urgent need for SARS-CoV-2 serologic tests to identify all infected individuals, irrespective of clinical symptoms, to conduct surveillance and implement strategies to contain spread. As the receptor-binding domain (RBD) of the spike protein is poorly conserved between SARS-CoVs and other pathogenic human coronaviruses, the RBD represents a promising antigen for detecting CoV-specific antibodies in people. Here, we use a large panel of human sera (63 SARS-CoV-2 patients and 71 control individuals) and hyperimmune sera from animals exposed to zoonotic CoVs to evaluate RBD’s performance as an antigen for reliable detection of SARS-CoV-2–specific antibodies. By day 9 after the onset of symptoms, the recombinant SARS-CoV-2 RBD antigen was highly sensitive (98%) and specific (100%) for antibodies induced by SARS-CoVs. We observed a strong correlation between levels of RBD-binding antibodies and SARS-CoV-2 neutralizing antibodies in patients. Our results, which reveal the early kinetics of SARS-CoV-2 antibody responses, support using the RBD antigen in serological diagnostic assays and RBD-specific antibody levels as a correlate of SARS-CoV-2 neutralizing antibodies in people.

Background & Aims Human norovirus infection is the leading cause of acute gastroenteritis. Genetic polymorphisms, mediated by the FUT2 gene (secretor enzyme), define strain susceptibility. Secretors express a diverse set of fucosylated histoblood group antigen carbohydrates (HBGA) on mucosal cells; nonsecretors (FUT2-/-) express a limited array of HBGAs. Thus, nonsecretors have less diverse norovirus strain infections, including resistance to the epidemiologically dominant GII.4 strains. Because future human norovirus vaccines will comprise GII.4 antigen and because secretor phenotype impacts GII.4 infection and immunity, nonsecretors may mimic young children immunologically in response to GII.4 vaccination, providing a needed model to study cross-protection in the context of limited pre-exposure. Methods By using specimens collected from the first characterized nonsecretor cohort naturally infected with GII.2 human norovirus, we evaluated the breadth of serologic immunity by surrogate neutralization assays, and cellular activation and cytokine production by flow cytometry. Results GII.2 infection resulted in broad antibody and cellular immunity activation that persisted for at least 30 days for T cells, monocytes, and dendritic cells, and for 180 days for blocking antibody. Multiple cellular lineages expressing interferon-γ and tumor necrosis factor-α dominated the response. Both T-cell and B-cell responses were cross-reactive with other GII strains, but not GI strains. To promote entry mechanisms, inclusion of bile acids was essential for GII.2 binding to nonsecretor HBGAs. Conclusions These data support development of within-genogroup, cross-reactive antibody and T-cell immunity, key outcomes that may provide the foundation for eliciting broad immune responses after GII.4 vaccination in individuals with limited GII.4 immunity, including young children.

Emergent strains of human norovirus seed pandemic waves of disease. These new strains have altered ligand binding and antigenicity characteristics. Study of viral variants isolated from immunosuppressed patients with long-term norovirus infection indicates that initial virus in vivo evolution occurs at the same antigenic sites as in pandemic strains. Here, cellular ligand binding and antigenicity of two co-circulating strains isolated from a patient with long-term norovirus infection were characterized. The isolated GII.4 viruses differed from previous strains and from each other at known blockade antibody epitopes. One strain had a unique sequence in epitope D, including loss of an insertion at residue 394, corresponding to a decreased relative affinity for carbohydrate ligands. Replacement of 394 with alanine or restoration of the contemporary strain epitope D consensus sequence STT improved ligand binding relative affinity. However, monoclonal antibody blockade of binding potency was only gained for the consensus sequence, not by the alanine insertion. In-depth study of unique changes in epitope D indicated that ligand binding, but not antibody blockade of ligand binding, is maintained despite sequence diversity, allowing escape from blockade antibodies without loss of capacity for binding cellular ligands. IMPORTANCE Human norovirus causes 20% of all acute gastroenteritis and 200,000 deaths per year, primarily in young children. Most epidemic and all pandemic waves of disease over the past 30 years have been caused by type GII.4 human norovirus strains. The capsid sequence of GII.4 strains is changing over time, resulting in viruses with altered ligand and antibody binding characteristics. The carbohydrate binding pocket of these strains does not vary over time. Here, utilizing unique viral sequences, we study how residues in GII.4 epitope D balance the dual roles of variable antibody binding site and cellular ligand binding stabilization domain, demonstrating that amino acid changes in epitope D can result in loss of antibody binding without ablating ligand binding. This flexibility in epitope D likely contributes to GII.4 strain persistence by both allowing escape from antibody-mediated herd immunity and maintenance of cellular ligand binding and infectivity.