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• Although Coxevac® is applied to sheep to prevent Coxiellosis the dose is unclear. • Little side effects occurred after vaccination with 1 and 2 ml Coxevac® in sheep. • Immunized sheep reacted first with an IgM and IgG Phase II response. • A vaccine volume of 1 ml seems sufficient to induce an immune response. • A third vaccination is necessary to stimulate the cellular immunity in naïve sheep. An inactivated Coxiella burnetii Phase I (PhI) vaccine (Coxevac®) is licensed in several European countries for goats and cattle to prevent coxiellosis. The vaccine is also applied to sheep, although detailed information about the ovine immune response and vaccine dose is missing. Eighteen gimmers from a C. burnetii unsuspected flock were randomly divided into three groups of six. Group 1 (Cox1) and 2 (Cox2) were vaccinated twice with 1 ml and 2 ml Coxevac®, respectively, three weeks apart (primary vaccination). The same procedure was applied with Cox3 (2 ml sodium chloride, control group). A third injection (booster) was performed after nine months. Potential side effects were determined by measuring the rectal body temperature and skin thickness at the injection site. Blood samples were collected to detect phase-specific IgM and IgG antibodies and interferon-ɣ (IFN-ɣ) release by immunofluorescence assay and ELISAs, respectively. Moreover, a cell infection neutralization assay determined the appearance of neutralizing sera. Body temperatures increased for one day post vaccination, and the skin swelled only slightly. Regardless of the vaccine volume, immunized sheep reacted first with an IgM and IgG PhII response. Ten weeks after the primary vaccination, IgG PhI antibodies predominated. Boosting eight months after primary vaccination resulted in a robust IgG PhI increase and strong IFN-ɣ response. In the vaccinated animals, the neutralizing effect is more widespread after the administration of 1 ml than after the treatment with 2 ml. In summary, differences between 1 and 2 ml Coxevac® are minor, and a vaccine volume of 1 ml seems to be sufficient. A booster after the primary vaccination is apparently necessary to stimulate the cell-mediated immune response in naïve sheep. [ABSTRACT FROM AUTHOR]

The interpretation for Zika virus serology results is challenging due to high antibody cross reactivity with other flaviviruses. This limits availability of reliable and accurate methods for serosurveillance studies to understand the disease burden. Therefore, we conducted study to harmonize anti-Zika IgG antibody detection assays with 1st WHO International Standard (16/352) and working standard (16/320) for anti-Zika virus antibody.Additionally, evaluated NuGenTMZIKA-IgG and NovaLisa®ZIKA virus IgG-Capture ELISA using a panel of 278 seraFurther, 106 samples positive for other-flavi viruses were taken for assessing cross-reactivity of the assay, all serums were further tested by Zika-PRNT. The results of this study indicates satisfactory performance of both the assays. Serological and neutralization assays were calibrated according to the international standards. This will help in understanding antibody dynamics in serosurveillance and vaccine studies. However the performance of the kits with possibilities of cross-reactivity will have to be verified by coupling ZIKV and DENV specific ELISA. [ABSTRACT FROM AUTHOR]

• Antibodies to spike and nucleocapsid antigens have research and clinical utility. • BioRad SARS-CoV-2 IgG assay incorporates both spike and nucleocapsid antigens. • Performance of BioRad SARS-CoV-2 IgG multiplex assay comparable to existing assays. • Combination of multiple antigens increases clinical sensitivity over single antigen. • Levels of RBD binding antibodies correlate well with neutralizing antibodies (FRNT). Detection of antibodies to multiple SARS-CoV-2 antigens in a single assay could increase diagnostic accuracy, differentiate vaccination from natural disease, and aid in retrospective exposure determination. Correlation of binding antibody assessment in clinical assays with neutralizing antibodies is needed to better understand the humoral response to SARS-CoV-2 infection and establish of correlates of protection. A cohort of 752 samples was used to assess specificity, sensitivity, and comparison to 6 other Conformitè Europëenne serologic assays for the BioRad SARS-CoV-2 IgG multiplex assay which measures receptor binding domain IgG (RBD), spike-S1 IgG (S1), spike-S2 IgG (S2), and nucleocapsid IgG (N). A subset of serial specimens from 14 patients was also tested for neutralizing antibodies (n = 61). Specificity for RBD and S1 IgG was 99.4% (n = 170) and 100% for S2 and N IgG (n = 170) in a cohort selected for probable interference. Overall assay concordance with other assays was >93% for IgG and total antibody assays and reached 100% sensitivity for clinical concordance at >14 days as a multiplex assay. RBD and S1 binding antibody positivity demonstrated 79–95% agreement with the presence of neutralizing antibodies. The BioRad SARS-CoV-2 IgG assay is comparable to existing assays, and achieved 100% sensitivity when all markers were included. The ability to measure antibodies against spike and nucleocapsid proteins simultaneously may be advantageous for complex clinical presentations, epidemiologic research, and in decisions regarding infection prevention strategies. Additional independent validations are needed to further determine binding antibody and neutralizing antibody correlations. [ABSTRACT FROM AUTHOR]

In order to meet the domestic urgent needs of evaluating the immunogenicity of vaccines and the potency testing of therapeutic antibody products against coronavirus disease 2019 (COVID-19), the first Chinese national standards for SARS-CoV-2 neutralizing antibody were established. The potency and stability of the candidate standards were determined by neutralization assay and accelerated degradation study. The stability studies showed that the standards were stable in the short-term. The collaborative study showed that the candidate standards could reduce the variations in neutralization titers between labs and thus improve comparability of neutralizing antibody measurements. Sample 22 has been approved by the Biological Product Reference Standards Sub-Committee of the National Drug Reference Standards Committee as the first Chinese National Standard for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) neutralizing antibody, with an assigned potency of 1,000 units per milliliter (U/ml). This standard will contribute to the standardized assessment of the quality and efficacy of vaccines and therapeutics for COVID-19 in China. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 infection and development of coronavirus disease 2019 presents a major health care challenge of global dimensions. Laboratory diagnostics of infected patients, and the assessment of immunity against severe acute respiratory syndrome coronavirus 2, presents a major cornerstone in handling the pandemic. Currently, there is an increase in demand for antibody testing and a large number of tests are already marketed or are in the late stage of development. However, the interpretation of test results depends on many variables and factors, including sensitivity, specificity, potential cross-reactivity and cross-protectivity, the diagnostic value of antibodies of different isotypes, and the use of antibody testing in identification of acutely ill patients or in epidemiological settings. In this article, the recently established COVID-19 Task Force of the German Society for Clinical Chemistry and Laboratory Medicine (DGKL) addresses these issues on the basis of currently available data sets in this rapidly moving field. [ABSTRACT FROM AUTHOR]

Abstract Arenaviruses cause several viral hemorrhagic fevers endemic to Africa and South America. The respective causative agents are classified as biosafety level (BSL) 4 pathogens. Unlike for most other BSL4 agents, for the New World arenavirus Junín virus (JUNV) both a highly effective vaccination (Candid#1) and a post-exposure treatment, based on convalescent plasma transfer, are available. In particular, neutralizing antibodies (nAbs) represent a key protective determinant in JUNV infection, which is supported by the correlation between successful passive antibody therapy and the levels of nAbs administered. Unfortunately, comparable resources for the management of other closely related arenavirus infections are not available. Given the significant challenges inherent in studying BSL4 pathogens, our goal was to first assess the suitability of a JUNV transcription and replication-competent virus-like particle (trVLP) system for measuring virus neutralization under BSL1/2 conditions. Indeed, we could show that infection with JUNV trVLPs is glycoprotein (GP) dependent, that trVLP input has a direct correlation to reporter readout, and that these trVLPs can be neutralized by human serum with kinetics similar to those obtained using authentic virus. These properties make trVLPs suitable for use as a proxy for virus in neutralization assays. Using this platform we then evaluated the potential of JUNV nAbs to cross-neutralize entry mediated by GPs from other arenaviruses using JUNV (strain Romero)-based trVLPs bearing GPs either from other JUNV strains, other closely related New World arenaviruses (e.g. Tacaribe, Machupo, Sabiá), or the distantly related Lassa virus. While nAbs against the JUNV vaccine strain are also active against a range of other JUNV strains, they appear to have little or no capacity to neutralize other arenavirus species, suggesting that therapy with whole plasma directed against another species is unlikely to be successful and that the targeted development of cross-specific monoclonal antibody-based resources is likely needed. Such efforts will be supported by the availability of this BSL1/2 screening platform which provides a rapid and easy means to characterize the potency and reactivity of anti-arenavirus neutralizing antibodies against a range of arenavirus species. Graphical abstract Image 1 Highlights • trVLP entry is dependent on the glycoprotein and displays a direct correlation to reporter signal. • trVLPs can be used as a proxy for virus to measure neutralizing antibody activity under reduced biosafety conditions (BSL1/2). • trVLP assays are quicker and less labor intensive to perform and allow an objective automated readout. • Glycoproteins can be exchanged on trVLPs (even between distantly related arenaviruses) allowing testing of cross-reactivity. • Sera from Candid#1 vaccinees show neutralization for multiple JUNV strains, but not for other closely related virus species. [ABSTRACT FROM AUTHOR]

Highlights • We successfully produced pseudotypes carrying the hemagglutinin (HA) subtype H5 or H7 influenza virus subtypes and lacZ as reporter gene. • The HA-pseudotype titers decreased and showed a significant statistical difference under various storage temperatures of storage and freeze-thaw cycles, but the reduction will likely not affect their use in serological techniques. • The titers remained were optimal for application in neutralization assays. Abstract Retroviral pseudotypes are broadly used as safe instruments to mimic the structure and surface of highly pathogenic viruses. They have been employed for the discovery of new drugs, as diagnostic tools in vaccine studies, and part of serological assays. Because of their widespread use in research and their potential as tools for quality control, it is important to know their shelf life, stability, and best storage conditions. In this study, we produced pseudotypes carrying the lacZ reporter gene and the hemagglutinin (HA) of avian influenza virus subtypes H5 and H7 to investigate their stability under various storage conditions. We produced pseudotypes with titers of approximately 106 RLU/mL, which decreased to 105–106 RLU/mL after short-term storage at 4 °C (up to 4 weeks). Stability was maintained after long-term storage at −20 °C (up to 12 months), even under storage variations such as freeze-thaw cycles. We conclude that, although the titers decreased by 1 log 10 under the different storage conditions, the remaining titers can be readily applicable in many techniques, such as neutralization assays. These findings show that large quantities of retroviral pseudotypes can be safely stored for short- or long-term use, allowing standardization and reduced variation in assays involving retroviral pseudotypes. [ABSTRACT FROM AUTHOR]

Acute hepatopancreatic necrosis disease (AHPND) has a significant economic impact on aquaculture worldwide. However, the molecular mechanism responsible for AHPND remains unknown. Our previous study demonstrated that the Litopenaeus vannamei fatty acid binding protein (LvFABP) interacted with the Vibrio parahaemolyticus toxin PirB, and that silencing LvFABP using RNA interference improved AHPND resistance in shrimp. Polyclonal antibodies against recombinant LvFABP and PirBvp toxin were produced to further elucidate the role of LvFABP in AHPND resistance. Immunofluorescence localization showed that LvFABP was abundant throughout the cytoplasm of the hepatopancreas cells, and a co-localization assay revealed that PirBvp directly interacted with LvFABP. Antibodies against LvFABP were able to partially block the binding of LvFABP to PirBvp in vitro and inhibited AHPND infection in vivo. Exogenous recombinant LvFABP had a partially neutralizing effect against AHPND and gave L. vannamei significant protection against infection. In addition, recombinant LvFABP treatment of L. vannamei significantly reduced the morphological damage to the shrimp following pathogen challenge. The transcriptomes of the L. vannamei hepatopancreas with and without V. parahaemolyticus infection after silencing of LvFABP by RNA interference were also explored. The results showed that AHPND infection could affect the health of L. vannamei via functional changes in carbohydrate metabolism, lipid metabolism, and transport and catabolism. Furthermore, silencing LvFABP could effectively protect metabolism, transport and catabolic homeostasis, thus enabling adaptation to further or prolonged AHPND infections. These findings provided a unique and important dataset that deepened our understanding of the pathological mechanism of AHPND and may help in the development of strategies to prevent and ameliorate the effects of bacterial diseases. • The Vibrio parahaemolyticus toxin PirB (PirBvp) directly interacted with LvFABP. • Blocking the interaction between LvFABP and PirBvp significantly reduced the occurrence of AHPND. • Carbohydrate metabolism, lipid metabolism, and transport and catabolism pathways responded significantly to AHPND. • Silencing LvFABP could effectively maintain metabolism, transport and catabolic homeostasis. [ABSTRACT FROM AUTHOR]

A licensed vaccine for respiratory syncytial virus (RSV) has yet to be developed, and a reliable and repeatable neutralizing assay is indispensable for vaccine development. Here, we demonstrated an optimized high-throughput RSV neutralization assay that utilizes a fluorescence plate reader (reader) as a substitute for flow cytometry to detect fluorescent signals in RSV-A2 mKate-infected cells. Furthermore, this study tested the influence of virus input and infectivity on the neutralizing assay and highlighted critical factors (together with a suggested protocol) for obtaining stable data using this assay. [ABSTRACT FROM AUTHOR]

Dengue virus infection elicits immune responses to multiple viral antigens including antibodies to dengue non-structural protein 1 (NS1) which are rapidly induced and detected within days of infection. The recombinant, live, attenuated, tetravalent dengue vaccine (CYD-TDV; Sanofi Pasteur) uses the yellow fever vaccine virus as a back-bone but expresses dengue virus pre-membrane and envelop proteins. Since CYD-TDV does not express dengue NS1, we evaluated the utility of dengue NS1-specific IgG antibodies as biomarkers of dengue exposure in CYD-TDV recipients and controls. We optimized and evaluated a quantitative anti-dengue NS1 IgG enzyme-linked immunosorbent assay (ELISA). Parameters assessed included: accuracy, dilutability/linearity, precision, limit of quantitation and specificity. The assay specificity was further evaluated using Japanese Encephalitis virus, West Nile virus, Yellow Fever virus or Zika virus positive sera samples collected following confirmed infection or vaccination. Receiver-operating-characteristics (ROC) curves as well as sensitivity and specificity for discriminating previous dengue exposure were assessed using 1250 reference samples. Overall, the anti-dengue NS1 IgG ELISA was able to discriminate previous dengue exposure from non-exposure before vaccination with CYD-TDV (ROC area under the curve > 0.9). Assessment of paired samples from 2511 vaccinated participants showed high overall agreement (93%) between pre-vaccination and post-vaccination dengue serostatus classification based on the anti-dengue NS1 IgG ELISA. However, misclassification of dengue serostatus was observed after vaccination likely due to a combination of asymptomatic dengue infections, assay variability and a modest effect of CYD-TDV on the anti-dengue NS1 IgG ELISA readout. [ABSTRACT FROM AUTHOR]

Background Autoimmune (type 1) diabetes (T1D) is a frequent chronic disease in children and adolescents globally. Gestational enterovirus (EV) infections have been associated with an increased risk for T1D in the offspring. We test the hypothesis that EV infections during the first trimester were associated with beta cell autoantibodies in mothers of children who developed islet autoantibodies before 7 years of age. Materials and methods Local registries were used to identify mothers to children born 2000–2007 who developed either beta cell autoantibodies or T1D during follow up. Serum samples from the first trimester were located in the Biobank. A total of 448 index mothers were identified and compared to 891 matched control mothers. EV-IgM was determined in a capture enzyme immunoassay. Beta cell autoantibodies were analyzed in standard radio binding assays. Results The frequency of EV-IgM in index mothers was 20% (89/448), which did not differ from the control mothers 20% (175/891) ( p  = 0.922). Index mothers had multiple beta cell autoantibodies more often than control mothers (p = 0.037). Beta cell autoantibodies were increased during the November–April winter months in index compared to control mothers (p = 0.022). The observed difference was possibly explained by the months of February-April (p = 0.014). Concomitant EV-IgM and beta cell autoantibodies tended to be more common among index compared to control mothers (p = 0.039). Conclusion EV-IgM during the first trimester may be associated with beta cell autoantibodies in mothers to children who developed either beta cell autoantibodies or T1D before 7 years of age. [ABSTRACT FROM AUTHOR]

Manipulation of the flavivirus genome to accommodate and express a heterologous gene of interest has become an attractive approach for gene delivery and the development of viral-vectored vaccines. However, due to the inherent genetic instability of the flavivirus genomes, the construction of recombinant viruses carrying a foreign gene could be problematic and heavily resistant. In this study, the possibility of the Japanese encephalitis virus (JEV) as a stable flavivirus vector for the expression of a foreign gene was assessed using reverse genetics. The full-length cDNA genome of genotype I (GI) JEV inherently possessed excellent stability and manipulability in a bacterial host, while mutations and deletions accumulated in the cDNA genomes of genotype Ⅲ (GⅢ) JEV strains. Using the GI JEV as backbones, we generate a panel of recombinant viruses expressing various foreign genes. All recombinant viruses exhibited excellent genetic stability and efficiently express foreign genes for at least ten serial passages in vitro. In application, a convenient, rapid and reliable image-based assay for neutralizing antibody testing and antiviral drug discovery was established with a mCherry-reporter recombinant virus (rBJ-mCherry). Meanwhile, the recombinant viruses expressing the antigens of the African swine fever virus (ASFV) or Classical swine fever virus (CSFV) could effectively induce antibody responses to the JEV vector and foreign antigens in a mouse vaccination model. Therefore, GI JEV strains could serve as viral vectors accommodating the expression of large foreign genes. • The infectious cDNA clones of GI JEV strains remain stable in E. coli. • GI JEV strains derived vectors can accommodate foreign gene expression. • GI JEV reporter viruses were generated for antiviral and neutralization assays. • GI JEV vaccine candidate was explored for combination vaccine development. [ABSTRACT FROM AUTHOR]

COVID-19 convalescent plasma (CCP) could improve the clinical outcome of COVID-19 patients when high-titer CCP is administered in early stages of disease. However, CCP donors have a risk profile like first-time donors, pathogen reduction treatment (PRT) may mitigate such risk but should not impact CCP quality. The current study aims to assess the impact of PRT-technologies available in Saudi Arabia on the neutralizing activity of CCP. and Methods: CCP was collected from eligible donors by plasmapheresis. The neutralization titer was determined with an in-house microneutralization assay (MNA) using a local SARS-CoV-2 clinical isolate. Selected units were split and subject to PRT with amotosalen/UVA (AS) or Riboflavin/UVB (RB) (pairwise side-by-side comparison) followed by a second MNA analysis. 51 CCP units were collected, 27 were included in the analysis reaching the minimum MNA titer of 1:40 (4 reached high titer (≥1:250)). 27 CCP units were treated with AS and 14 with RB, the median MNA pre-treatment titer was 1:80 (1:40–640). The impact of AS and RB PRT on CCP neutralizing activity was not significantly different, nor in the total analysis neither in the pairwise comparison (94.6 vs 96.4 % retention, p > 0.05). No correlation of titer and blood group was observed, but a trend for increasing MNA titer with donor age, choosing donors with an age > 45 years would increase the number of high-titer CCP donors. The difference in impact of AS and RB on CCP MNA titer was below the limit of detection of the assay (0.5-fold). [ABSTRACT FROM AUTHOR]

Development of vaccines against highly pathogenic viruses that could also be used as agents of bioterrorism is both a public health issue and a national security priority. Methods that can quantify neutralizing antibodies will likely be crucial in demonstrating vaccine effectiveness, as most licensed viral vaccines are effective due to their capacity to elicit neutralizing antibodies. Assays to determine whether antibodies are neutralizing traditionally involve infectious virus, and the assay most commonly used is the plaque-reduction neutralization test (PRNT). However, when the virus is highly pathogenic, this assay must be done under the appropriate level of containment; for tier one select agents, such as Ebola virus (EBOV), it is performed under Biological Safety Level 4 (BSL-4) conditions. Developing high-throughput neutralization assays for these viruses that can be done in standard BSL-2 laboratories should facilitate vaccine development. Our approach is to use a replication-competent hybrid virus whose genome carries the envelope gene from the pathogenic virus on the genetic backbone of a non-pathogenic virus, such as vesicular stomatitis virus (VSV). We have generated hybrid VSVs carrying the envelope genes for several species of ebolavirus. The readout for infectivity is a one-step reverse transcriptase quantitative PCR (RT-qPCR), an approach that we have used for other viruses that allows robustness and adaptability to automation. Using this method, we have shown that neutralization can be assessed within 6–16 h after infection. Importantly, the titers obtained in our assay with two characterized antibodies were in agreement with titers obtained in other assays. Finally, although in this paper we describe the VSV platform to quantify neutralizing antibodies to ebolaviruses, the platform should be directly applicable to any virus whose envelope is compatible with VSV biology. [ABSTRACT FROM AUTHOR]

Despite the serious public health impact of Crimean-Congo hemorrhagic fever (CCHF), the efficacy of antivirals targeting the causative agent, CCHF virus (CCHFV), remains debatable. Neutralizing monoclonal antibodies (MAbs) targeting the CCHFV glycoprotein Gc have been reported to protect mice against challenge with the prototype CCHFV strain, IbAr10200. However, due to extensive sequence diversity of CCHFV glycoproteins, it is unknown whether these MAbs neutralize other CCHFV strains. We initially used a CCHF virus-like particle (VLP) system to generate 11 VLP moieties, each possessing a glycoprotein from a genetically diverse CCHFV strain isolated in either Africa, Asia, the Middle East, or southeastern Europe. We used these VLPs in biosafety level 2 conditions to efficiently screen MAb cross-neutralization potency. Of the 16 MAbs tested, 3 (8A1, 11E7, and 30F7) demonstrated cross-neutralization activity with most CCHF VLPs, with 8A1 neutralizing all VLPs tested. Although binding studies suggest that none of the MAbs compete for the same epitope, combining 11E7, 30F7, or both 11E7 and 30F7 with 8A1 had no additive effect on increasing neutralization in this system. To confirm our findings from the VLP system, the 3 MAbs capable of strain cross-neutralization were confirmed to effectively neutralize 5 diverse CCHFV strains in vitro. Passaging CCHFV strains in the presence of sub-neutralizing concentrations of MAbs did not generate escape mutants resistant to subsequent neutralization. This study demonstrates the utility of the VLP system for screening neutralizing MAbs against multiple CCHFV strains, and provides the first evidence that a single MAb can effectively neutralize a number of diverse CCHFV strains in vitro, which may lead to development of future CCHF therapeutics. [ABSTRACT FROM AUTHOR]

Monoclonal antibody (mAb) therapy is one of the most promising immunotherapies that have shown the potential to prevent or neutralize the effects of COVID-19 in patients at very early stages, with a few formulations recently approved by the European and American medicine agencies. However, a main bottleneck for their general implementation resides in the time-consuming, laborious, and highly-specialized techniques employed for the manufacturing and assessing of these therapies, excessively increasing their prices and delaying their administration to the patients. We propose a biomimetic nanoplasmonic biosensor as a novel analytical technique for the screening and evaluation of COVID-19 mAb therapies in a simpler, faster, and reliable manner. By creating an artificial cell membrane on the plasmonic sensor surface, our label-free sensing approach enables real-time monitoring of virus-cell interactions as well as direct analysis of antibody blocking effects in only 15 min assay time. We have achieved detection limits in the 102 TCID50/mL range for the study of SARS-CoV-2 viruses, which allows to perform neutralization assays by only employing a low-volume sample with common viral loads. We have demonstrated the accuracy of the biosensor for the evaluation of two different neutralizing antibodies targeting both Delta and Omicron variants of SARS-CoV-2, with half maximal inhibitory concentrations (IC 50) determined in the ng/mL range. Our user-friendly and reliable technology could be employed in biomedical and pharmaceutical laboratories to accelerate, cheapen, and simplify the development of effective immunotherapies for COVID-19 and other serious infectious diseases or cancer. [ABSTRACT FROM AUTHOR]

Serological assays provide an indirect route for the recognition of infectious agents via the detection of antibodies against the infectious agent of interest within serum. Serological assays for equine influenza A virus can be applied for different purposes: diagnosing infections; subtyping isolates; surveillance of circulating strains; and to evaluate the efficacy of vaccines before they reach the market. Haemagglutination inhibition (HI) and single radial haemolysis (SRH) assays are most commonly used in the equine field. This review outlines how both these assays together with virus neutralization (VN) and ELISA are performed, interpreted and applied for the control of equine influenza, giving the limitations and advantages of each. The pseudotyped virus neutralization assay (PVNA) is also discussed as a promising prospect for the future of equine influenza virus serology. [ABSTRACT FROM AUTHOR]

Background The current outbreak of Ebola Virus Disease in West Africa is caused by a new variant of Ebola virus (EBOV) named Makona 2014, whose sequence differs 3% from isolates from Central Africa such as Mayinga 1976 EBOV. The specificity and kinetics of the neutralizing antibody response induced by the circulating Makona EBOV has not been thoroughly studied. Methods We have used a lentiviral EBOV-glycoprotein (GP)-pseudotyped infection assay to measure Makona-GP and Mayinga-GP specific neutralizing activity of plasma from three convalescent Ebola Virus Disease patients from the current EBOV outbreak at 2, 3, 4 and 9 months post-infection. Total anti-EBOV GP IgG was measured by a commercial ELISA assay. Findings In convalescent Ebola Virus Disease patients, Makona-GP-specific neutralizing titers increased from 2 months (mean IC 50 1/59), 3 months (IC 50 1/212), 4 months (IC 50 1/239) and up to 9 months (IC 50 1/268) post-infection. Neutralizing activity of plasma from the three convalescent Ebola Virus Disease patients was more vigorous against the current Makona-GP pseudotyped EBOV variant than against Mayinga-GP pseudotyped EBOV and this difference was observed at each time point tested: Mayinga vs Makona mean IC 50 fold = 4.92 at 2 months post-infection, 2.89 fold at 3 months post-infection, 2.23 at 4 months post-infection and 2.98 at 9 months post-infection (all differences p < 0.01). Total level of IgG against EBOV-GP did not evolve significantly during the follow up. Discussion In convalescent Ebola Virus Disease patients, EBOV-GP specific neutralizing activity increases over time, at least up to 9 months post-infection, which suggests that active affinity maturation of antibodies takes place long after clinical recovery. EBOV-GP specific neutralizing response is significantly higher against Makona EBOV circulating in West Africa than against the variants included in the currently approved vaccines. Correlates of protection for EBOV vaccines have not been completely established and the relevance of a lower neutralizing activity in convalescent plasma from the current outbreak against one of the EBOV-GPs contained in the vaccines in terms of its potential efficacy does not necessarily preclude its efficacy. However, this observation highlights the concern regarding the natural diversity of EBOV and its subsequent challenge for diagnosis, therapy and vaccine design. EBOV-GP neutralizing activity varies considerably over time in convalescent Ebola Virus Disease patients. Titering of convalescent blood products would be desirable to standardize and evaluate their potential therapeutic value. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) specific neutralizing and enhancing antibodies play crucial roles in dengue disease prevention and pathogenesis. DENV reporters are gaining popularity in the evaluation of these antibodies; their accessibility and acceptance may improve with more efficient production systems and indications of their antigenic equivalence to the wild-type virus. This study aimed to generate a replication competent luciferase-secreting DENV reporter (LucDENV2) and evaluate its feasibility in neutralizing and infection-enhancing antibody assays in comparison with wild-type DENV2, strain 16681, and a luciferase-secreting, single-round infectious DENV2 reporter (LucSIP). LucDENV2 replicated to similarly high levels as that of the parent 16681 virus in a commonly used mosquito cell line. LucDENV2 was neutralized in an antibody concentration-dependent manner by a monoclonal antibody specific to the flavivirus fusion loop and two antibodies specific to the E domain III, which closely resembled the neutralization patterns employing the LucSIP and wild-type DENV2. Parallel analysis of LucDENV2 and wild-type DENV2 revealed good agreement between the luciferase-based and focus-based neutralization and enhancement assays in a 96-well microplate format when employed against a set of clinical sera, suggesting comparable antigenic properties of LucDENV2 with those of the parent virus. The high-titer, replication competent, luciferase-secreting DENV reporter presented here should be a useful tool for fast and reliable quantitation of neutralizing and infection-enhancing antibodies in populations living in DENV-endemic areas. • High-titer, replication competent LucDENV2 generated in widely used mosquito cells. • LucDENV2, LucSIP and wild-type DENV2 similarly neutralized by anti-E antibodies. • Feasibility of LucDENV2 used in LRNT assay was shown with 30 DENV2 clinical sera. • Comparable antibody-mediated infection-enhancement of LucDENV2 and wild-type DENV2. [ABSTRACT FROM AUTHOR]

• A rapid Elispot-based neutralization assay against Coxsackievirus B1 (CVB1) was successfully developed. • The CVB1 Nt-Elispot is objective, highly sensitive, efficient, and high-throughput. • The CVB1 Nt-Elispot could be applied in the screening of anti-CVB1 neutralizing monoclonal antibodies. • The CVB1 Nt-Elispot could be used to detect NAb levels against CVB1 in large cohorts. Coxsackievirus B1 (CVB1) is a major pathogen that causes viral myocarditis and aseptic meningitis and is implicated as a cause of type 1 diabetes mellitus. The rapid detection of neutralizing antibodies can help in the prevention and diagnosis of viral infection. The traditional cytopathic effect (CPE)-based neutralization assay (Nt-CPE) is time-consuming and labor-intensive. In this study, an efficient neutralization test based on an enzyme-linked immunospot assay and a monoclonal antibody 2E6 against CVB1 (Nt-Elispot) was developed. In this optimal Nt-Elispot, a multiplicity of infection (MOI) of 1 per well was set as the infection dose, and an incubation time of 18 hours was selected as the checkpoint. Compared with Nt-CPE, Nt-Elispot significantly shortened the detection period and displayed a good correlation with it. This established CVB1 Nt-Elispot could be applied to efficiently screen neutralizing antibodies and evaluate the level of NAb against CVB1 in large cohorts. [ABSTRACT FROM AUTHOR]

Highlights: [•] Highly sensitive neutralization test based on high-content cell-by-cell screening. [•] Detects neutralizing antibodies within the first week after infection of an animal. [•] Takes less than 24h to complete. [•] Excludes operator bias by automated data acquisition and analysis. [•] Dose estimation by non-linear regression using a 4-parameter log-logistic model. [ABSTRACT FROM AUTHOR]

• Two immune-based methods (ICM and cell-based ELISA) are adapted to IPKMs. • This aids the quantification of infection of PRRSV field strains. • ICM and cell-based ELISA also offer comparable sensitivity. • ICM and cell-based ELISA are adequate for quantifying neutralizing activity. Porcine reproductive and respiratory syndrome virus (PRRSV) causes a serious infectious disease in pigs in farms worldwide. Neutralizing antibody titer is an effective index for evaluating immunity to PRRSV; however, PRRSV has different neutralizing cross-reactivity between strains. Therefore, quantitative measurement of neutralizing antibody titers against field PRRSV strains would be required to evaluate whether neutralizing antibodies in pigs could possess neutralizing activity against individual or multiple strains. Immune-based methods, such as image cytometry (ICM) and cell-based enzyme-linked immune sorbent assay (ELISA), are quantitative and can be used to evaluate many samples. Using immortalized porcine kidney macrophages (IPKMs), which are highly susceptible to infection from field PRRSV-2 strains compared with other cell lines, immune-based methods could enable the evaluation of the neutralizing activity of porcine serum against field strains of PRRSV-2 that are difficult to isolate in conventional cells. In summary, we adapted two methods, namely ICM and cell-based ELISA, to IPKMs for quantitative neutralizing antibody titer measurements. Two immune-based methods using IPKMs are adequate for quantifying neutralizing activity of porcine serum against PRRSV-2, including field strains. [ABSTRACT FROM AUTHOR]

Highlights: [•] Two ELISA assays are analyzed as correlates of immunity after influenza vaccination. [•] Antibodies against unfolded recombinant and against native hemagglutinin are detected. [•] Results are compared to microneutralization titers. [•] Healthy individuals and patients after kidney transplantation are investigated. [•] Both ELISA assays are correlated with microneutralization titers and can serve as markers of protection. [ABSTRACT FROM AUTHOR]

Abstract: Background: Human enterovirus 71 (HEV71) is a common cause of severe outbreaks of hand-foot- and mouth disease, aseptic meningitis and encephalitis in Asian populations but has not caused such epidemics in all populations. Objectives: To analyze the frequency of HEV71 in the background childhood population in Finland by screening in stool and serum samples and by measuring neutralizing antibodies against HEV71 in serum and to compare the genetic relationship of virus strains detected in asymptomatic children and those causing severe illness in Finland to the strains found in other countries. Study design: 4185 stool samples and 5686 serum samples were collected and clinical symptoms recorded from children who were observed from birth. Additional stool samples were available from four children hospitalized due to EV71 infection. Samples were screened for the presence of RNA of human enteroviruses using RT-PCR and HEV71 amplicons were identified by sequencing. Phylogenetic analyses were carried out to study genetic relationships between different virus strains. Neutralizing antibodies against HEV71 were screened from 522 children. Results: A total of 0.3% of stool samples and two serum samples from healthy children were positive for HEV71 genome. 1.6% of the children had neutralizing antibodies against HEV71. Most infections were asymptomatic or mild in contrast to the clear symptoms in the children hospitalized due to HEV71. All viruses were C strains. Conclusions: HEV71 is circulating in Finland but it is rare. No clear difference was seen between strains circulating in the Finnish background population and those found in hospitalized patients or those causing severe outbreaks worldwide. [Copyright &y& Elsevier]

Abstract: Background: Human metapneumovirus (HMPV) is a common cause of respiratory diseases in persons of all ages. Because of its slow replication and weak cytopathic effect in cultured cells, conventional neutralization assays for HMPV require around one week for completion. Objectives: The purpose of this study is to establish a rapid neutralization assay based on a recombinant virus expressing Renilla luciferase (Rluc). Study design: A recombinant HMPV expressing both Rluc and green fluorescent protein (GFP) was created by reverse genetics method. Two-fold serial dilutions of human 23 sera were made in a 96-well plate and incubated with 50pfu/well of the recombinant virus at 4°C for 1h. The mixtures were then transferred to LLC-MK2 cells in a 96-well plate, incubated for 2h, and replaced with trypsin-free fresh media. After incubation at 32°C for 24h, the cells were lysed and measured for Rluc activity. The neutralization titer was defined as the reciprocal of the highest serum dilution that resulted in 50% reduction of Rluc activity. Results: The novel assay could be completed within 24h and eliminated the requirement of trypsin supporting multistep replication in cultured cells, as well as laborious processes including the plaque assay with immunostaining. Neutralization titers correlated well with those determined by a GFP-based assay previously developed. Conclusions: The neutralization assay based on Rluc activity is the fastest and the most straightforward of all previous assays, and may be available for high throughput screening of neutralizing antibodies. [Copyright &y& Elsevier]

Abstract: A real-time cell analysis (RTCA) system based on cell-substrate electric impedance technology was used to monitor cytopathic effects (CPE) in Vero cell cultures infected with West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) at infectious doses ranging from 101 to 106 plaque forming units (PFU) of virus. A kinetic parameter characterizing virus-induced CPE, CIT50 or the time to 50% decrease in cell impedance, was inversely proportional to virus infectious dose. In WNV-infected cells, the onset and rate of CPE was earlier and faster than in SLEV-infected cells, which was consistent with viral cytolytic activity. A mathematical model simulating impedance-based CPE kinetic curves indicated that the replication rate of WNV was about 3 times faster than SLEV. The RTCA system also was used for quantifying the level of cell protection by specific neutralizing antibodies against WNV and SLEV. The onset of WNV or SLEV-induced CPE was delayed in the presence of specific anti-sera, and this delay in the CIT50 was well correlated with the titer of the neutralizing antibody as measured independently by plaque reduction neutralization tests (PRNT). The RTCA system provided a high throughput and quantitative method for real-time monitoring viral growth in cell culture and its inhibition by neutralizing antibodies. [Copyright &y& Elsevier]

Abstract: The hemagglutination inhibition (HI) assay has been the main method used to investigate immune responses to vaccination against influenza H1N1 (2009) virus. However microneutralization tests (MNT) have been shown to be more sensitive and more specific. In this study, the three methods of choice: (i) the HI assay, (ii) an ELISA-based conventional MNT and (iii) a colorimetric MNT in terms of their ability to detect antibody responses in serum pairs collected from 43 healthy individuals before and 21 days after vaccination were compared. The colorimetric MNT was established yielding intra- and inter-run imprecisions of 7.5% and 12.4%, respectively. Testing of antisera to seasonal influenza viruses demonstrated the assay to be specific for antibodies to influenza H1N1 (2009) virus. A good correlation between the three methods was found, being highest for the ELISA-MNT and the colorimetric MNT (r =0.714 for geometric mean titers (GMT) and r =0.695 for titer increases). Similar rates of fourfold titer increases were detected: 95.3% in the ELISA-MNT vs. 93.0% in colorimetric MNT and 95.3% in HI assay. The ELISA-based MNT demonstrated the highest titer range leading to the highest postvaccination GMT and the highest titer increase (>50-fold). The lowest GMTs were measured with the HI assay, while the colormetric MNT detected the highest GMT in prevaccination sera. Taken together, similar seroconversion rates were obtained with the three assays. The ELISA-MNT appeared to be the best method to compare absolute pre- and postvaccination GMTs. The colorimetric MNT, being less labour-intensive than the ELISA-MNT, seems to be a suitable tool in vaccination studies. [ABSTRACT FROM AUTHOR]

Abstract: Influenza vaccine immunogenicity is commonly assessed by determining hemagglutination inhibition (HAI) titers in serum samples. HAI titers have been used to predict vaccine efficacy, but this often fails when live attenuated vaccines are evaluated, because it does not encompass all immune mediators of protection. Although antibodies that inhibit viral neuraminidase (NA) also contribute to protection against disease, there is currently no routine assessment of NA inhibition titers. A serological method with the capacity to measure functional inhibition of both HA and NA would be valuable. We developed a high-throughput virus neutralization assay that uses viral NA activity to quantify influenza replication (the AVINA assay), and showed its capacity to identify antivirals with a broad range of target specificities. In this report we demonstrate that antibodies with specificity for either HA or NA are detected in this assay, whereas a commonly used virus neutralization assay only detects those with HA-specificity. We also compared human responses to seasonal influenza vaccines measured by HAI, micro-neutralization, NA inhibition and AVINA assays. The response rates to both trivalent inactivated and live attenuated vaccines were greater when measured by the AVINA than the other assays, reflecting the dual antigen reactivity and increased sensitivity of the assay. The potential of this single assay to predict protection against influenza-induced tachypnea was demonstrated in vaccinated cotton rats. The AVINA assay is therefore a practical, comprehensive method to determine influenza vaccine immunogenicity and potential efficacy. [Copyright &y& Elsevier]

Abstract: Specific, effective and rapid neutralization assays are crucial for the development of an HIV vaccine based on the stimulation of neutralizing antibodies and the development of such an assay for the human immunodeficiency virus-2 (HIV-2) is described. Virus neutralization was measured as the reduction of provirus integration using a duplex real-time PCR with high efficiency (99.4%). This PCR uses primers and a probe specific for the proviral LTR. Amplification and quantitative analysis of the cellular GAPDH gene was carried out in parallel to control for toxic or growth-inhibitory components in the sera. The neutralization assay was used to screen sera from 23 HIV-2 infected patients. 21 sera were able to neutralize HIV-260415K, 20 sera neutralized HIV-27312A and 7 sera cross-neutralized HIV-1 IIIB. In contrast, when 14 of these sera were tested in parallel with a conventional neutralization assay based on a p27Gag capture ELISA, only one was found to neutralize HIV-260415K and 11 to neutralize HIV-27312A compared with 12 and 13 sera respectively using the PCR-based assay. [Copyright &y& Elsevier]

• H5 and H7-pseudotypes: safe and specific alternative to serological tools. • Hemagglutinin pseudotypes as potential novel tools for quality control of influenza vaccines. • New approaches to subtype-specific detection of influenza H5 and H7 antibodies. Influenza is a relevant problem for public and animal health, with a significant economic impact. In recent years, outbreaks of avian influenza virus have resulted in devastating losses in the poultry industry worldwide, and although its transmission to humans is very rare, there is always a potential risk for an even more severe outbreak. Currently, vaccination is considered the most effective tool for the control and prevention of influenza infections in both humans and animals. The maintenance of animal welfare and the successful implementation of animal health programs depend on the timely administration of vaccines, which must comply with quality specifications indicated by health authorities; for example, the capability to ensure a minimum antibody titer. The production of viral antigens used in these tests can pose a biosafety risk, and some viral strains can be difficult to grow. Therefore, new biotechnological alternatives are required to overcome these disadvantages. In this study, we produced pseudotypes carrying H5 and H7 hemagglutinins from lowly and highly pathogenic avian influenza viruses. These pseudotypes were used in neutralization assays to detect neutralizing antibodies in avian sera, which were confirmed positive by inhibition of the hemagglutination test. Our results showed that the pseudotype neutralization assay is a viable alternative for the detection of neutralizing antibodies, by demonstrating subtype specificity and requiring reduced biosafety requirements. Therefore, it represents a versatile platform that can facilitate technology transfer protocols between laboratories, and an immediate application in serological tools for quality control of veterinary vaccines against avian influenza. [ABSTRACT FROM AUTHOR]

• The clinical sera showed higher neutralizing activity against Sabin 1 and 3 pseudovirus. • The clinical sera showed higher neutralizing activity against wild-type 2 pseudovirus. • The pseudovirus-based neutralizing assay could substitute the live virus assay. The cost-effectiveness of the Sabin inactivated poliovirus vaccine derived from the Sabin strains (S-IPV) and its reduced biosecurity risks during its manufacture make it the vaccine of choice over the IPVs derived from wild-type polioviruses. However, it is difficult to evaluate whether S-IPVs can achieve wild-type poliovirus containment in China, making its development there less attractive. To facilitate the development and adoption of S-IPVs in China, the aim of this study was to develop an alternative neutralizing assay using either a polio pseudovirus derived from a Sabin strain (S-pNA) or one derived from a wild-type strain (w-pNA) to replace the conventional neutralizing assay which uses live polioviruses. A total of 100 sera were collected from children immunized with an oral poliovirus vaccine and their antibody titers were assessed by both the S-pNA and w-pNA. The results showed that this method was feasible for the quantification of neutralizing antibody activities in the sera of the vaccinated individuals. The Wilcoxon signed-rank sum test indicated that the neutralizing antibody titers obtained against the Sabin strains were higher than those obtained with the wild-type strains for types 1 and 3, while for type 2, the titers against the wild-type strains were higher than those against the Sabin strains (p < 0.001 for all three types). It is hoped that this assay could be used to assess whether immune sera by the S-IPV possess adequate neutralizing capacity against both attenuated and wild-type poliovirus strains. [ABSTRACT FROM AUTHOR]

Abstract: Ideal methods to monitor HPV neutralizing antibodies induced by vaccination have not been established yet. Here, we evaluated systemic and cervical antibody levels induced by HPV16/18 AS04-adjuvanted vaccine (GlaxoSmithKline Biologicals) using a secreted alkaline phosphatase neutralization assay (SEAP-NA) and enzyme-linked immunosorbent assay (ELISA). Serum and cervical secretions from 50 vaccinated women were used to assess (1) overall assay reproducibility; (2) inter-assay and inter-specimen correlation; (3) correlations between month 1 and month 12 titers. Strong correlations between SEAP-NA and ELISA were observed (serum anti-HPV16/18, ρ =0.91/0.85; cervix anti-HPV16/18, ρ =0.84/0.89). Systemic and cervical antibody measures also correlated well (ρ range: 0.64–0.75); except at mid-cycle (ρ range: 0.28–0.65). Correlations between antibody levels at 1 and 12 months following the start of vaccination were poor (ρ range: 0.16–0.38). In conclusion, HPV16/18 VLP-based ELISA is a reliable and valid method to monitor anti-HPV16/18 neutralizing potential for the first year following vaccination; however, additional studies will be required to better define the effects of the time on cycle and patterns of antibody response over time following vaccination. [Copyright &y& Elsevier]

Abstract: A fluorescent quantitative PCR (FQ-PCR) assay utilizing SYBR green I dye is described for quantitation of white spot syndrome virus (WSSV) particles isolated from infected crayfish, Cambarus clarkii. For this assay, a primer set was designed which amplifies, with high efficiency and specificity, a 129bp target sequence within ORF167 of the WSSV genome. Conveniently, WSSV particles can be added into the FQ-PCR assay with a simple and convenient method to release its DNA. To establish the basis for an in vitro neutralization test, primary cultures of shrimp cells were challenged with WSSV that had been incubated with a polyclonal anti-WSSV serum or with control proteins. The number of WSSV particles released from the cells after these treatments were assayed by FQ-PCR. This test may serve as a method to screen monoclonal antibody pools or recombinant antibody pools for neutralizing activity prior to in vivo animal experiments. [Copyright &y& Elsevier]

Abstract: Human papillomavirus 16 virus-like particle (HPV16 VLP) vaccines expressed in Saccharomyces cerevisiae are under Phase III trial and are expected to be on the market in the near future. We have established a convenient and economical system for the prophylactic study of vaccines derived from HPV16 VLPs, and neutralization tests to standardize HPV serological methodology as a measure of validation. To purify HPV16 VLPs, yeast cells expressing HPV16 L1 protein were cultured and purified on a small scale by ultracentrifugation and size-exclusion and cation-exchange chromatography using open columns. The highly purified HPV16 L1 protein was identified by SDS-PAGE and Western blotting, and electron microscopic analysis confirmed that they self-assembled into VLPs. To test the efficacy of the purified VLPs as a vaccine and their ability to induce humoral immunity, we performed ELISA assays and observed a significant increase in the titer of anti-HPV16 VLPs antibodies in the sera of immunized mice. High anti-HPV16 neutralizing titers were found in the sera of vaccinated mice, as measured by a SEAP-based pseudovirus neutralization assay. These results would be useful in the evaluation of the immunogenicity of HPV vaccine candidates, and provide an international reference standard for HPV serological methods. [Copyright &y& Elsevier]

Abstract: An attempt was made to neutralize white spot syndrome virus of shrimp using the antiserum against recombinant VP28 protein of WSSV. The dilutions of virus stock ranged from 1×101 to 1×108 were used to determine the dilution resulting in 90–100% mortality in Penaeus monodon by intramuscular injection of WSSV. A constant amount of WSSV (at 1×0104) was incubated with various antiserum concentrations and injected into shrimp. No shrimp died in the negative control injected only with NTE buffer. The shrimp in the positive control, which were injected with WSSV only showed 100% mortality at 10-day post infection (d p.i.). The WSSV preincubated with preimmune serum caused 100% mortality of shrimp at 10 d p.i. None of the shrimp injected with the preimmune serum only died. When the WSSV was preincubated with two or one time diluted anti r-VP28, shrimp mortality was 100% and 75%, respectively, at 50 d p.i. apparently the anti r-VP28 at these dilutions is not able to neutralize the WSSV. When the WSSV was preincubated with undiluted anti r-VP28, none of the shrimp died, indicating that WSSV can be neutralized by the anti r-VP28 in a dose-dependent manner. [Copyright &y& Elsevier]

Abstract: Accurate assessment of neutralizing antibody activities is important either for patients infected with Severe Acute Respiratory Syndrome (SARS) or for animals and volunteers immunized with the experimental vaccines against the SARS associated coronavirus (SCV). However, the current assay based on the cytopathic effect (CPE) which has been frequently cited in literature has several limitations. The CPE assay relies on the visual observation on the damage of SCV infected target cells under a microscope. It is subjected to observer variations and it is difficult to generate a quantitative determination of neutralizing activities based on the level of CPE. In the current study, we established the utility of two additional assays to measure the neutralizing activities against SCV: the plaque reduction (PR) and the neutral red staining (NRS) assays. The PR assay described in this study was modified from the traditional viral plaque reduction assay by using an improved crystal staining method to achieve better plague formation in SCV infected Vero E6 cells. The NRS neutralization assay was adopted from a similar system used for detecting neutralizing antibody responses against human immunodeficiency virus type 1 (HIV-1). In this assay, the protective effect of neutralizing antibodies was determined by the cell viability which is measured by the uptake of neutral red dye at A540. The neutralizing antibody titers can be easily determined with either of the two new assays. In this report, we described the utility of these two new neutralization assays in measuring the neutralizing activities against SCV infection from rabbit sera immunized with various forms of spike protein of SCV. [Copyright &y& Elsevier]