A new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus.

Dengue is a rapidly emerging, mosquito-borne viral infection, with an estimated 400 million infections occurring annually. To gain insight into dengue immunity, we characterized 145 human monoclonal antibodies (mAbs) and identified a previously unknown epitope, the envelope dimer epitope (EDE), that bridges two envelope protein subunits that make up the 90 repeating dimers on the mature virion. The mAbs to EDE were broadly reactive across the dengue serocomplex and fully neutralized virus produced in either insect cells or primary human cells, with 50% neutralization in the low picomolar range. Our results provide a path to a subunit vaccine against dengue virus and have implications for the design and monitoring of future vaccine trials in which the induction of antibody to the EDE should be prioritized.

Comparing the performance of dengue virus IgG and IgG-capture enzyme-linked immunosorbent assays in seroprevalence study.

BACKGROUND: Dengue virus (DENV) is the leading cause of arboviral diseases in humans worldwide. Currently Dengvaxia, the first dengue vaccine licensed in 20 countries, was recommended for DENV seropositive individuals aged 9-45 years. Studying dengue seroprevalence can improve our understanding of the epidemiology and transmission dynamics of DENV, and facilitate future intervention strategies and assessment of vaccine efficacy. Several DENV envelope protein-based serological tests including IgG and IgG-capture enzyme-linked immunosorbent assays (ELISAs) have been employed in seroprevalence studies. Previously DENV IgG-capture ELISA was reported to distinguish primary and secondary DENV infections during early convalescence, however, its performance over time and in seroprevalence study remains understudied. METHODS: In this study, we used well-documented neutralization test- or reverse-transcription-polymerase-chain reaction-confirmed serum/plasma samples including DENV-naïve, primary and secondary DENV, primary West Nile virus, primary Zika virus, and Zika with previous DENV infection panels to compare the performance of three ELISAs. RESULTS: The sensitivity of the InBios IgG ELISA was higher than that of InBios IgG-capture and SD IgG-capture ELISAs. The sensitivity of IgG-capture ELISAs was higher for secondary than primary DENV infection panel. Within the secondary DENV infection panel, the sensitivity of InBios IgG-capture ELISA decreased from 77.8% at < 6 months to 41.7% at 1-1.5 years, 28.6% at 2-15 years and 0% at > 20 years (p < 0.001, Cochran-Armitage test for trend), whereas that of IgG ELISA remains 100%. A similar trend was observed for SD IgG-capture ELISA. CONCLUSIONS: Our findings demonstrate higher sensitivity of DENV IgG ELISA than IgG-capture ELISA in seroprevalence study and interpretation of DENV IgG-capture ELISA should take sampling time and primary or secondary DENV infection into consideration.

Identification of Anti-Premembrane Antibody as a Serocomplex-Specific Marker To Discriminate Zika, Dengue, and West Nile Virus Infections.

Although transmission of Zika virus (ZIKV) in the Americas has greatly declined since late 2017, recent reports of reduced risks of symptomatic Zika by prior dengue virus (DENV) infection and increased risks of severe dengue disease by previous ZIKV or DENV infection underscore a critical need for serological tests that can discriminate past ZIKV, DENV, and/or other flavivirus infections and improve our understanding of the immune interactions between these viruses and vaccine strategy in endemic regions. As serological tests for ZIKV primarily focus on envelope (E) and nonstructural protein 1 (NS1), antibodies to other ZIKV proteins have not been explored. Here, we employed Western blot analysis using antigens of 6 flaviviruses from 3 serocomplexes to investigate antibody responses following reverse transcription-PCR (RT-PCR)-confirmed ZIKV infection. Panels of 20 primary ZIKV and 20 ZIKV with previous DENV infection recognized E proteins of all 6 flaviviruses and the NS1 protein of ZIKV with some cross-reactivity to DENV. While the primary ZIKV panel recognized only the premembrane (prM) protein of ZIKV, the ZIKV with previous DENV panel recognized both ZIKV and DENV prM proteins. Analysis of antibody responses following 42 DENV and 18 West Nile virus infections revealed similar patterns of recognition by anti-E and anti-NS1 antibodies, whereas both panels recognized the prM protein of the homologous serocomplex but not others. The specificity was further supported by analysis of sequential samples. Together, these findings suggest that anti-prM antibody is a flavivirus serocomplex-specific marker and can be used to delineate current and past flavivirus infections in endemic areas. IMPORTANCE Despite a decline in Zika virus (ZIKV) transmission since late 2017, questions regarding its surveillance, potential reemergence, and interactions with other flaviviruses in regions where it is endemic remain unanswered. Recent studies have reported reduced risks of symptomatic Zika by prior dengue virus (DENV) infection and increased risks of severe dengue disease by previous ZIKV or DENV infection, highlighting a need for better serological tests to discriminate past ZIKV, DENV, and/or other flavivirus infections and improved understanding of the immune interactions and vaccine strategy for these viruses. As most serological tests for ZIKV focused on envelope and nonstructural protein 1, antibodies to other ZIKV proteins, including potentially specific antibodies, remain understudied. We employed Western blot analysis using antigens of 6 flaviviruses to study antibody responses following well-documented ZIKV, DENV, and West Nile virus infections and identified anti-premembrane antibody as a flavivirus serocomplex-specific marker to delineate current and past flavivirus infections in areas where flaviviruses are endemic.

Combination of Nonstructural Protein 1-Based Enzyme-Linked Immunosorbent Assays Can Detect and Distinguish Various Dengue Virus and Zika Virus Infections.

The recent outbreaks of Zika virus (ZIKV) and associated birth defects in regions of dengue virus (DENV) endemicity emphasize the need for sensitive and specific serodiagnostic tests. We reported previously that enzyme-linked immunosorbent assays (ELISAs) based on the nonstructural protein 1 (NS1) of DENV serotype 1 (DENV1) and ZIKV can distinguish primary DENV1, secondary DENV, and ZIKV infections. Whether ELISAs based on NS1 proteins of other DENV serotypes can discriminate various DENV and ZIKV infections remains unknown. We herein developed DENV2, DENV3, and DENV4 NS1 IgG ELISAs to test convalescent- and postconvalescent-phase samples from reverse transcription-PCR-confirmed cases, including 25 primary DENV1, 24 primary DENV2, 10 primary DENV3, 67 secondary DENV, 36 primary West Nile virus, 38 primary ZIKV, and 35 ZIKV with previous DENV infections as well as 55 flavivirus-naive samples. Each ELISA detected primary DENV infection with a sensitivity of 100% for the same serotype and 23.8% to 100% for different serotypes. IgG ELISA using a mixture of DENV1-4 NS1 proteins detected different primary and secondary DENV infections with a sensitivity of 95.6% and specificity of 89.5%. The ZIKV NS1 IgG ELISA detected ZIKV infection with a sensitivity of 100% and specificity of 82.9%. On the basis of the relative optical density ratio, the combination of DENV1-4 and ZIKV NS1 IgG ELISAs distinguished ZIKV with previous DENV and secondary DENV infections with a sensitivity of 91.7% to 94.1% and specificity of 87.0% to 95.0%. These findings have important applications to serodiagnosis, serosurveillance, and monitoring of both DENV and ZIKV infections in regions of endemicity.

Potent Neutralizing Human Monoclonal Antibodies Preferentially Target Mature Dengue Virus Particles: Implication for Novel Strategy for Dengue Vaccine.

The four serotypes of dengue virus (DENV) cause the most important mosquito-borne viral disease in humans. The envelope (E) protein is the major target of neutralizing antibodies and contains 3 domains (domain I [DI], DII, and DIII). Recent studies reported that human monoclonal antibodies (MAbs) recognizing DIII, the D1/DII hinge, the E-dimer epitope, or a quaternary epitope involving DI/DII/DIII are more potently neutralizing than those recognizing the fusion loop (FL) of DII. Due to inefficient cleavage of the premembrane protein, DENV suspensions consist of a mixture of mature, immature, and partially immature particles. We investigated the neutralization and binding of 22 human MAbs to DENV serotype 1 (DENV1) virions with differential maturation status. Compared with FL MAbs, DIII, DI/DII hinge, and E-dimer epitope MAbs showed higher maximum binding and avidity to mature particles relative to immature particles; this feature may contribute to the strong neutralizing potency of such MAbs. FL-specific MAbs required 57 to 87% occupancy on mature particles to achieve half-maximal neutralization (NT50), whereas the potently neutralizing MAbs achieved NT50 states at 20 to 38% occupancy. Analysis of the MAb repertoire and polyclonal sera from patients with primary DENV1 infection supports the immunodominance of cross-reactive anti-E antibodies over type-specific antibodies. After depletion with viral particles from a heterologous DENV serotype, the type-specific neutralizing antibodies remained and showed binding features shared by potent neutralizing MAbs. Taken together, these findings suggest that the use of homogeneous mature DENV particles as an immunogen may induce more potent neutralizing antibodies against DENV than the use of immature or mixed particles.IMPORTANCE With an estimated 390 million infections per year, the four serotypes of dengue virus (DENV) cause the most important mosquito-borne viral disease in humans. The dengue vaccine Dengvaxia was licensed; however, its low efficacy among dengue-naive individuals and increased risk of causing severe dengue in children highlight the need for a better understanding of the role of human antibodies in immunity against DENV. DENV suspensions contain mature, immature, and partially immature particles. We investigated the binding of 22 human monoclonal antibodies (MAbs) to the DENV envelope protein on particles with different maturation states. Potently neutralizing MAbs had higher relative maximum binding and avidity to mature particles than weakly neutralizing MAbs. This was supported by analysis of MAb repertoires and polyclonal sera from patients with primary DENV infection. Together, these findings suggest that mature particles may be the optimal form of presentation of the envelope protein to induce more potent neutralizing antibodies against DENV.

Sustained Specific and Cross-Reactive T Cell Responses to Zika and Dengue Virus NS3 in West Africa.

Recent studies on the role of T cells in Zika virus (ZIKV) infection have shown that T cell responses to Asian ZIKV infection are important for protection, and that previous dengue virus (DENV) exposure amplifies the protective T cell response to Asian ZIKV. Human T cell responses to African ZIKV infection, however, remain unexplored. Here, we utilized the modified anthrax toxin delivery system to develop a flavivirus enzyme-linked immunosorbent spot (ELISPOT) assay. Using human ZIKV and DENV samples from Senegal, West Africa, our results demonstrate specific and cross-reactive T cell responses to nonstructural protein 3 (NS3). Specifically, we found that T cell responses to NS3 protease are ZIKV and DENV specific, but responses to NS3 helicase are cross-reactive. Sequential sample analyses revealed immune responses sustained many years after infection. These results have important implications for African ZIKV/DENV vaccine development, as well as for potential flavivirus diagnostics based on T cell responses.IMPORTANCE The recent Zika virus (ZIKV) epidemic in Latin America and the associated congenital microcephaly and Guillain-Barré syndrome have raised questions as to why we have not recognized these distinct clinical diseases in Africa. The human immunologic response to ZIKV and related flaviviruses in Africa represents a research gap that may shed light on the mechanisms contributing to protection. The goal of our study was to develop an inexpensive assay to detect and characterize the T cell response to African ZIKV and DENV. Our data show long-term specific and cross-reactive human immune responses against African ZIKV and DENV, suggesting the usefulness of a diagnostic based on the T cell response. Additionally, we show that prior flavivirus exposure influences the magnitude of the T cell response. The identification of immune responses to African ZIKV and DENV is of relevance to vaccine development.

Use of Urea Wash ELISA to Distinguish Zika and Dengue Virus Infections.

Serologic testing remains crucial for Zika virus diagnosis. We found that urea wash in a Zika virus nonstructural protein 1 IgG ELISA distinguishes secondary dengue virus infection from Zika virus infection with previous dengue (sensitivity 87.5%, specificity 93.8%). This test will aid serodiagnosis, serosurveillance, and monitoring of Zika complications in dengue-endemic regions.

Distinguishing Secondary Dengue Virus Infection From Zika Virus Infection With Previous Dengue by a Combination of 3 Simple Serological Tests.

BACKGROUND: The explosive spread of Zika virus (ZIKV) and associated microcephaly present an urgent need for sensitive and specific serodiagnostic tests, particularly for pregnant women in dengue virus (DENV)-endemic regions. Recent reports of enhanced ZIKV replication by dengue-immune sera have raised concerns about the role of previous DENV infection on the risk and severity of microcephaly and other ZIKV complications. METHODS: Enzyme-linked immunosorbent assays (ELISAs) based on ZIKV and DENV nonstructural protein 1 (NS1) were established to test acute, convalescent phase, and post-convalescent phase serum/plasma samples from reverse-transcription polymerase chain reaction-confirmed cases including 20 primary ZIKV, 25 ZIKV with previous DENV, 58 secondary DENV, and 16 primary DENV1 infections. RESULTS: ZIKV-NS1 immunoglobulin M (IgM) and immunoglobulin G (IgG) ELISAs combined can detect ZIKV infection with a sensitivity of 95% and specificity of 66.7%. The ZIKV-NS1 IgG cross-reactivity by samples from secondary DENV infection cases ranged from 66.7% to 28.1% (within 1 month to 1-2 years post-illness, respectively). Addition of DENV1-NS1 IgG ELISA can distinguish primary ZIKV infection; the ratio of absorbance of ZIKV-NS1 to DENV1-NS1 IgG ELISA can distinguish ZIKV with previous DENV and secondary DENV infections with a sensitivity of 87.5% and specificity of 81.3%. These findings were supported by analysis of sequential samples. CONCLUSIONS: An algorithm for ZIKV serodiagnosis based on 3 simple ELISAs is proposed to distinguish primary ZIKV, ZIKV with previous DENV, and secondary DENV infections; this could be applied to serodiagnosis for ZIKV, serosurveillance, and monitoring ZIKV infection during pregnancy to understand the epidemiology, pathogenesis, and complications of ZIKV in dengue-endemic regions.

Complexity of Neutralizing Antibodies against Multiple Dengue Virus Serotypes after Heterotypic Immunization and Secondary Infection Revealed by In-Depth Analysis of Cross-Reactive Antibodies.

UNLABELLED: The four serotypes of dengue virus (DENV) cause the most important and rapidly emerging arboviral diseases in humans. The recent phase 2b and 3 studies of a tetravalent dengue vaccine reported a moderate efficacy despite the presence of neutralizing antibodies, highlighting the need for a better understanding of neutralizing antibodies in polyclonal human sera. Certain type-specific (TS) antibodies were recently discovered to account for the monotypic neutralizing activity and protection after primary DENV infection. The nature of neutralizing antibodies after secondary DENV infection remains largely unknown. In this study, we examined sera from 10 vaccinees with well-documented exposure to first and second DENV serotypes through heterotypic immunization with live-attenuated vaccines. Higher serum IgG avidities to both exposed and nonexposed serotypes were found after secondary immunization than after primary immunization. Using a two-step depletion protocol to remove different anti-envelope antibodies, including group-reactive (GR) and complex-reactive (CR) antibodies separately, we found GR and CR antibodies together contributed to more than 50% of neutralizing activities against multiple serotypes after secondary immunization. Similar findings were demonstrated in patients after secondary infection. Anti-envelope antibodies recognizing previously exposed serotypes consisted of a large proportion of GR antibodies, CR antibodies, and a small proportion of TS antibodies, whereas those recognizing nonexposed serotypes consisted of GRand CR antibodies. These findings have implications for sequential heterotypic immunization or primary immunization of DENV-primed individuals as alternative strategies for DENV vaccination. The complexity of neutralizing antibodies after secondary infection provides new insights into the difficulty of their application as surrogates of protection. IMPORTANCE: The four serotypes of dengue virus (DENV) are the leading cause of arboviral diseases in humans. Despite the presence of neutralizing antibodies, a moderate efficacy was recently reported in phase 2b and 3 trials of a dengue vaccine; a better understanding of neutralizing antibodies in polyclonal human sera is urgently needed.We studied vaccinees who received heterotypic immunization of live-attenuated vaccines, as they were known to have received the first and second DENV serotype exposures.We found anti-envelope antibodies consist of group-reactive (GR), complex-reactive (CR), and type-specific (TS) antibodies, and that both GR and CR antibodies contribute significantly to multitypic neutralizing activities after secondary DENV immunization. These findings have implications for alternative strategies for DENV vaccination. Certain TS antibodies were recently discovered to contribute to the monotypic neutralizing activity and protection after primary DENV infection; our findings of the complexity of neutralizing activities after secondary immunization/infection provide new insights for neutralizing antibodies as surrogates of protection.

High-avidity and potently neutralizing cross-reactive human monoclonal antibodies derived from secondary dengue virus infection.

The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies (Abs) and vaccine development. Previous studies of human dengue-immune sera reported that a significant proportion of anti-E Abs, known as group-reactive (GR) Abs, were cross-reactive to all four DENV serotypes and to one or more other flaviviruses. Based on studies of mouse anti-E monoclonal antibodies (MAbs), GR MAbs were nonneutralizing or weakly neutralizing compared with type-specific MAbs; a GR response was thus not regarded as important for vaccine strategy. We investigated the epitopes, binding avidities, and neutralization potencies of 32 human GR anti-E MAbs. In addition to fusion loop (FL) residues in E protein domain II, human GR MAbs recognized an epitope involving both FL and bc loop residues in domain II. The neutralization potencies and binding avidities of GR MAbs derived from secondary DENV infection were stronger than those derived from primary infection. GR MAbs derived from primary DENV infection primarily blocked attachment, whereas those derived from secondary infection blocked DENV postattachment. Analysis of the repertoire of anti-E MAbs derived from patients with primary DENV infection revealed that the majority were GR, low-avidity, and weakly neutralizing MAbs, whereas those from secondary infection were primarily GR, high-avidity, and potently neutralizing MAbs. Our findings suggest that the weakly neutralizing GR anti-E Abs generated from primary DENV infection become potently neutralizing MAbs against the four serotypes after secondary infection. The observation that the dengue immune status of the host affects the quality of the cross-reactive Abs generated has implications for new strategies for DENV vaccination.

Determining the Time of Booster Dose Based on the Half-Life and Neutralization Titers against SARS-CoV-2 Variants of Concern in Fully Vaccinated Individuals.

Although mRNA-based COVID-19 vaccines reduce the risk of severe disease, hospitalization and death, vaccine effectiveness (VE) against infection and disease from variants of concern (VOC) wanes over time. Neutralizing antibodies (NAb) are surrogates of protection and are enhanced by a booster dose, but their kinetics and durability remain understudied. Current recommendation of a booster dose does not consider the existing NAb in each individual. Here, we investigated 50% neutralization (NT50) titers against VOC among COVID-19-naive participants receiving the Moderna (n = 26) or Pfizer (n = 25) vaccine for up to 7 months following the second dose, and determined their half-lives. We found that the time it took for NT50 titers to decline to 24, equivalent to 50% inhibitory dilution of 10 international units/mL, was longer in the Moderna (325/324/235/274 days for the D614G/alpha/beta/delta variants) group than in the Pfizer (253/252/174/226 days) group, which may account for the slower decline in VE of the Moderna vaccine observed in real-world settings and supports our hypothesis that measuring the NT50 titers against VOC, together with information on NAb half-lives, can be used to dictate the time of booster vaccination. Our study provides a framework to determine the optimal time of a booster dose against VOC at the individual level. In response to future VOC with high morbidity and mortality, a quick evaluation of NAb half-lives using longitudinal serum samples from clinical trials or research programs of different primary-series vaccinations and/or one or two boosters could provide references for determining the time of booster in different individuals. IMPORTANCE Despite improved understanding of the biology of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the evolutionary trajectory of the virus is uncertain, and the concern of future antigenically distinct variants remains. Current recommendations for a COVID-19 vaccine booster dose are primarily based on neutralization capacity, effectiveness against circulating variants of concern (VOC), and other host factors. We hypothesized that measuring neutralizing antibody titers against SARS-CoV-2 VOC together with half-life information can be used to dictate the time of booster vaccination. Through detailed analysis of neutralizing antibodies against VOC among COVID-19-naive vaccinees receiving either of two mRNA vaccines, we found that the time it took for 50% neutralization titers to decline to a reference level of protection was longer in the Moderna than in the Pfizer group, which supports our hypothesis. In response to future VOC with potentially high morbidity and mortality, our proof-of-concept study provides a framework to determine the optimal time of a booster dose at the individual level.

The helical domains of the stem region of dengue virus envelope protein are involved in both virus assembly and entry.

The envelope (E) of dengue virus (DENV) is a determinant of tropism and virulence. At the C terminus of E protein, there is a stem region containing two amphipathic α-helical domains (EH1 and EH2) and a stretch of conserved sequences in between. The crystal structure of E protein at the postfusion state suggested the involvement of the stem during the fusion; however, the critical domains or residues involved remain unknown. Site-directed mutagenesis was carried out to replace each of the stem residues at the hydrophobic face with an alanine or proline in a DENV serotype 4 (DENV4) precursor membrane (prM)/E expression construct. Most of the 15 proline mutations at either EH1 or EH2 severely affected the assembly of virus-like particles (VLPs). Radioimmunoprecipitation and membrane flotation assays revealed that EH1 mutations primarily affect prM-E heterodimerization and EH2 mutations affect the membrane binding of the stem. Introducing four proline mutations at either EH1 or EH2 into a DENV2 replicon packaging system greatly affects assembly and entry. Moreover, introducing these mutations into a DENV2 infectious clone confirmed the impairment in assembly and infectivity. Sequencing analysis of adaptive mutations in passage 5 viruses revealed a change to a leucine or wild-type residue at the original site, suggesting the importance of maintaining the helical structure. Collectively, these findings suggest that the EH1 and EH2 domains are involved in both assembly and entry steps of the DENV replication cycle; this feature, together with the high degree of sequence conservation, suggests that the stem region is a potential target of antiviral strategies.

Obtention of Dengue Virus Membrane Proteins and Role for Virus Assembly.

The four serotypes of dengue virus (DENV), belonging to the genus Flavivirus in the family Flaviviridae, are the leading cause of arboviral diseases in humans. The clinical presentations range from dengue fever to dengue hemorrhagic fever and dengue shock syndrome. Despite decades of efforts on developing intervention strategies against DENV, there is no licensed antiviral, and safe and effective vaccines remain challenging. Similar to other flaviviruses, the assembly of DENV particles occurs in the membranes derived from endoplasmic reticulum; immature virions bud into the lumen followed by maturation in the trans-Golgi and transport through the secretary pathway. A unique feature of flavivirus replication is the production of small and slowly sedimenting subviral particles, known as virus-like particles (VLPs). Co-expression of premembrane (prM) and envelope (E) proteins can generate recombinant VLPs, which are biophysically and antigenically similar to infectious virions and have been employed to study the function of prM and E proteins, assembly, serodiagnostic antigens, and vaccine candidates. Previously, we have developed several assays including sucrose cushion ultracentrifugation, sucrose gradient ultracentrifugation, membrane flotation, subcellular fractionation, and glycosidase digestion assay to exploit the interaction between DENV prM and E proteins, membrane association, subcellular localization, glycosylation pattern, and assembly of VLPs and replicon particles. The information derived from these assays have implications to further our understanding of DENV assembly, replication cycle, intervention strategies, and pathogenesis.

The length of and nonhydrophobic residues in the transmembrane domain of dengue virus envelope protein are critical for its retention and assembly in the endoplasmic reticulum.

The morphogenesis of many enveloped viruses, in which viral nucleocapsid complex interacts with envelope (E) protein, is known to take place at various sites along the secretory pathway. How viral E protein retains in a particular intracellular organelle for assembly remains incompletely understood. In this study, we investigated determinants in the E protein of dengue virus (DENV) for its retention and assembly in the endoplasmic reticulum (ER). A chimeric experiment between CD4 and DENV precursor membrane/E constructs suggested that the transmembrane domain (TMD) of E protein contains an ER retention signal. Substitutions of three nonhydrophobic residues at the N terminus of the first helix (T1) and at either the N or C terminus of the second helix of the TMD with three hydrophobic residues, as well as an increase in the length of T1, led to the release of chimeric CD4 and E protein from the ER, suggesting that short length and certain nonhydrophobic residues of the TMD are critical for ER retention. The analysis of enveloped viruses assembled at the plasma membrane and of those assembled in the Golgi complex and ER revealed a trend of decreasing length and increasing nonhydrophobic residues of the TMD of E proteins. Taken together, these findings support a TMD-dependent sorting for viral E proteins along the secretory pathway. Moreover, similar mutations introduced into the TMD of DENV E protein resulted in the increased production of virus-like particles (VLPs), suggesting that modifications of TMD facilitate VLP production and have implications for utilizing flaviviral VLPs as serodiagnostic antigens and vaccine candidates.

A real-time and high-throughput neutralization test based on SARS-CoV-2 pseudovirus containing monomeric infrared fluorescent protein as reporter.

Neutralizing antibodies to SARS-CoV-2 have been shown to correlate with protection in animals and humans, disease severity, survival, and vaccine efficacy. With the ongoing large-scale vaccination in different countries and continuous surge of new variants of global concerns, a convenient, cost-effective and high-throughput neutralization test is urgently needed. Conventional SARS-CoV-2 neutralization test is tedious, time-consuming and requires a biosafety level 3 laboratory. Despite recent reports of neutralizations using different pseudoviruses with a luciferase or green fluorescent protein reporter, the laborious steps, inter-assay variability or high background limit their high-throughput potential. In this study we generated lentivirus-based pseudoviruses containing a monomeric infrared fluorescent protein reporter to develop neutralization assays. Similar tropism, infection kinetics and mechanism of entry through receptor-mediated endocytosis were found in the three pseudoviruses generated. Compared with pseudovirus D614, pseudovirus with D614G mutation had decreased shedding and higher density of S1 protein present on particles. The 50% neutralization titers to pseudoviruses D614 or D614G correlated with the plaque reduction neutralization titers to live SARS-CoV-2. The turn-around time of 48-72 h, minimal autofluorescence, one-step image quantification, expandable to 384-well, sequential readouts and dual quantifications by flow cytometry support its high-throughput and versatile applications at a non-reference and biosafety level 2 laboratory, in particular for assessing the neutralization sensitivity of new variants by sera from natural infection or different vaccinations during our fight against the pandemic.

Enzyme-linked immunosorbent assays using virus-like particles containing mutations of conserved residues on envelope protein can distinguish three flavivirus infections.

The recent outbreaks of Zika virus (ZIKV) in flavivirus-endemic regions highlight the need for sensitive and specific serological tests. Previously we and others reported key fusion loop (FL) residues and/or BC loop (BCL) residues on dengue virus (DENV) envelope protein recognized by flavivirus cross-reactive human monoclonal antibodies and polyclonal sera. To improve ZIKV serodiagnosis, we employed wild type (WT) and FL or FL/BCL mutant virus-like particles (VLP) of ZIKV, DENV1 and West Nile virus (WNV) in enzyme linked immunosorbent assays (ELISA), and tested convalescent-phase serum or plasma samples from reverse-transcription PCR-confirmed cases with different ZIKV, DENV and WNV infections. For IgG ELISA, ZIKV WT-VLP had a sensitivity of 100% and specificity of 52.9%, which was improved to 83.3% by FL/BCL mutant VLP and 92.2% by the ratio of relative optical density of mutant to WT VLP. Similarly, DENV1 and WNV WT-VLP had a sensitivity/specificity of 100%/70.0% and 100%/56.3%, respectively; the specificity was improved to 93.3% and 83.0% by FL mutant VLP. For IgM ELISA, ZIKV, DENV1 and WNV WT-VLP had a specificity of 96.4%, 92.3% and 91.4%, respectively, for primary infection; the specificity was improved to 93.7-99.3% by FL or FL/BCL mutant VLP. An algorithm based on a combination of mutant and WT-VLP IgG ELISA is proposed to discriminate primary ZIKV, DENV and WNV infections as well as secondary DENV and ZIKV infection with previous DENV infections; this could be a powerful tool to better understand the seroprevalence and pathogenesis of ZIKV in regions where multiple flaviviruses co-circulate.

Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II.

The antibody response to the envelope (E) glycoprotein of dengue virus (DENV) is known to play a critical role in both protection from and enhancement of disease, especially after primary infection. However, the relative amounts of homologous and heterologous anti-E antibodies and their epitopes remain unclear. In this study, we examined the antibody responses to E protein as well as to precursor membrane (PrM), capsid, and nonstructural protein 1 (NS1) of four serotypes of DENV by Western blot analysis of DENV serotype 2-infected patients with different disease severity and immune status during an outbreak in southern Taiwan in 2002. Based on the early-convalescent-phase sera tested, the rates of antibody responses to PrM and NS1 proteins were significantly higher in patients with secondary infection than in those with primary infection. A blocking experiment and neutralization assay showed that more than 90% of anti-E antibodies after primary infection were cross-reactive and nonneutralizing against heterologous serotypes and that only a minor proportion were type specific, which may account for the type-specific neutralization activity. Moreover, the E-binding activity in sera of 10 patients with primary infection was greatly reduced by amino acid replacements of three fusion loop residues, tryptophan at position 101, leucine at position 107, and phenylalanine at position 108, but not by replacements of those outside the fusion loop of domain II, suggesting that the predominantly cross-reactive anti-E antibodies recognized epitopes involving the highly conserved residues at the fusion loop of domain II. These findings have implications for our understanding of the pathogenesis of dengue and for the future design of subunit vaccine against DENV as well.