Pseudoparticle Neutralization Research Articles

Cross-Reactive Antibody Responses to Coronaviruses Elicited by SARS-CoV-2 Infection or Vaccination.

The newly emerged SARS-CoV-2 possesses shared antigenic epitopes with other human coronaviruses. We investigated if COVID-19 vaccination or SARS-CoV-2 infection may boost cross-reactive antibodies to other human coronaviruses. Prevaccination and postvaccination sera from SARS-CoV-2 naïve healthy subjects who received three doses of the mRNA vaccine (BioNTech, BNT) or the inactivated vaccine (CoronaVac, CV) were used to monitor the level of cross-reactive antibodies raised against other human coronaviruses by enzyme-linked immunosorbent assay. In comparison, convalescent sera from COVID-19 patients with or without prior vaccination history were also tested. Pseudoparticle neutralization assay was performed to detect neutralization antibody against MERS-CoV. Among SARS-CoV-2 infection-naïve subjects, BNT or CV significantly increased the anti-S2 antibodies against Betacoronaviruses (OC43 and MERS-CoV) but not Alphacoronaviruses (229E). The prevaccination antibody response to the common cold human coronaviruses did not negatively impact the postvaccination antibody response to SARS-CoV-2. Cross-reactive antibodies that binds to the S2 protein of MERS-CoV were similarly detected from the convalescent sera of COVID-19 patients with or without vaccination history. However, these anti-S2 antibodies do not possess neutralizing activity in MERS-CoV pseudoparticle neutralization tests. Our results suggest that SARS-CoV-2 infection or vaccination may potentially modulate population immune landscape against previously exposed or novel human coronaviruses. The findings have implications for future sero-epidemiological studies on MERS-CoV.

Effect of Different Adjuvants on the Longevity and Strength of Humoral and Cellular Immune Responses to the HCV Envelope Glycoproteins.

Infection by Hepatitis C virus (HCV) can lead to liver cirrhosis/hepatocellular carcinoma and remains a major cause of serious disease morbidity and mortality worldwide. However, current treatment regimens remain inaccessible to most patients, particularly in developing countries, and, therefore, the development of a novel vaccine capable of protecting subjects from chronic infection by HCV could greatly reduce the rates of HCV infection, subsequent liver pathogenesis, and in some cases death. Herein, we evaluated two different semi-synthetic archaeosome formulations as an adjuvant to the E1/E2 HCV envelope protein in a murine model and compared antigen-specific humoral (levels of anti-E1/E2 IgG and HCV pseudoparticle neutralization) and cellular responses (numbers of antigen-specific cytokine-producing T cells) to those generated with adjuvant formulations composed of mimetics of commercial adjuvants including a squalene oil-in-water emulsion, aluminum hydroxide/monophosphoryl lipid A (MPLA) and liposome/MPLA/QS-21. In addition, we measured the longevity of these responses, tracking humoral, and cellular responses up to 6 months following vaccination. Overall, we show that the strength and longevity of anti-HCV responses can be influenced by adjuvant selection. In particular, a simple admixed sulfated S-lactosylarchaeol (SLA) archaeosome formulation generated strong levels of HCV neutralizing antibodies and polyfunctional antigen-specific CD4 T cells producing multiple cytokines such as IFN-γ, TNF-α, and IL-2. While liposome/MPLA/QS-21 as adjuvant generated superior cellular responses, the SLA E1/E2 admixed formulation was superior or equivalent to the other tested formulations in all immune parameters tested.

Transmissibility of MERS-CoV Infection in Closed Setting, Riyadh, Saudi Arabia, 2015.

To investigate a cluster of Middle East respiratory syndrome (MERS) cases in a women-only dormitory in Riyadh, Saudi Arabia, in October 2015, we collected epidemiologic information, nasopharyngeal/oropharyngeal swab samples, and blood samples from 828 residents during November 2015 and December 2015–January 2016. We found confirmed infection for 19 (8 by reverse transcription PCR and 11 by serologic testing). Infection attack rates varied (2.7%–32.3%) by dormitory building. No deaths occurred. Independent risk factors for infection were direct contact with a confirmed case-patient and sharing a room with a confirmed case-patient; a protective factor was having an air conditioner in the bedroom. For 9 women from whom a second serum sample was collected, antibodies remained detectable at titers >1:20 by pseudoparticle neutralization tests (n = 8) and 90% plaque-reduction neutralization tests (n = 2). In closed high-contact settings, MERS coronavirus was highly infectious and pathogenicity was relatively low.

Middle East respiratory coronavirus (MERS-CoV) spike (S) protein vesicular stomatitis virus pseudoparticle neutralization assays offer a reliable alternative to the conventional neutralization assay in human seroepidemiological studies.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel zoonotic coronavirus that was identified in 2012. MERS-CoV infection in humans can result in an acute, severe respiratory disease and in some cases multi-organ failure; the global mortality rate is approximately 35 %. The MERS-CoV spike (S) protein is a major target for neutralizing antibodies in infected patients. The MERS-CoV microneutralization test (MNt) is the gold standard method for demonstrating prior infection. However, this method requires the use of live MERS-CoV in biosafety level 3 (BSL-3) containment. The present work describes the generation and validation of S protein-bearing vesicular stomatitis virus (VSV) pseudotype particles (VSV-MERS-CoV-S) in which the VSV glycoprotein G gene has been replaced by the luciferase reporter gene, followed by the establishment of a pseudoparticle-based neutralization test to detect MERS-CoV neutralizing antibodies under BSL-2 conditions. Using a panel of human sera from confirmed MERS-CoV patients, the VSV-MERS-CoV particle neutralization assay produced results that were highly comparable to those of the microneutralization test using live MERS-CoV. The results suggest that the VSV-MERS-CoV-S pseudotype neutralization assay offers a highly specific, sensitive and safer alternative method to detect MERS-CoV neutralizing antibodies in human sera.

A53 MERS-CoV in East African dromedary camels

Human Middle East respiratory syndrome is a zoonotic respiratory disease caused by Middle East respiratory syndrome coronavirus (MERS-CoV) originating from camels in the Arabian Peninsula. While there are a large number of camels in East Africa, often traded to the Arabian Peninsula, no autochthonous human MERS-CoV case is reported in East Africa. Furthermore, there is limited information of MERS-CoV in East Africa. In this study, MERS-CoV in dromedary camels from Ethiopia was detected using RT-qPCR. Next-generation sequencing was used to obtain the full genome of MERS-CoV. MERS-CoV antibodies were also detected through MERS-spike pseudoparticle neutralization assay. Phylogenetic analysis of full-genome sequences and spike-genome antibodies indicates that MERS-CoV in East Africa is genetically distinct from those in the Arabian Peninsula. The results from this study show that MERS-CoV circulating in dromedary camels in East Africa are genetically distinct from those in the Arabian Peninsula. Further studies are needed to evaluate the risk of zoonotic transmission in East Africa.

Circulating CXCR3+ Tfh cells positively correlate with neutralizing antibody responses in HCV-infected patients

Circulating T follicular helper (cTfh) cells have been identified as counterparts of germinal center Tfh (GC Tfh) cells in humans and can support T-dependent B cell maturation and antibody production in vitro. However, the role of cTfh cells in neutralizing antibody (nAb) responses in HCV infection remains unclear. Here, we characterized the phenotype and function of cTfh cells and demonstrated the associations of cTfh cells and their subsets with nAb responses in HCV infection. A total of 38 HCV-infected individuals and 28 healthy controls were enrolled from a pool of injection drug users. The frequency and function of blood Tfh cells were analyzed by flow cytometry. The titers and breadths of serum nAbs were measured using HCV pseudo-particle neutralization assays. Herein, we report several key observations. First, HCV infection skewed cTfh toward CXCR3+ cTfh cell differentiation. Second, the frequency of CXCR3+ cTfh cells positively correlated with HCV nAb titers and breadths. Third, CXCR3+ cTfh cells showed higher expression of Tfh-associated molecules (PD-1, ICOS, IL-21, Bcl-6) compared with CXCR3− cTfh cells from individuals with HCV infection. Coculture of cTfh cells and autologous memory B cells in vitro indicated that CXCR3+ cTfh cells show a superior ability to support HCV E2-specific B cell expansion compared with CXCR3− cTfh cells from individuals with HCV infection. HCV infection skews cTfh cells toward CXCR3-biased Tfh cell differentiation, which positively correlates with the magnitude and breadth of the HCV nAb response. It is our hope that these findings will provide insights for the rational design of a nAb-based HCV vaccine.

Elevated LAG-3 on CD4+ T cells negatively correlates with neutralizing antibody response during HCV infection

Lymphocyte activation gene-3 (LAG-3), an inhibitory molecule, which has been shown co-expressed with multiple inhibitory receptors on CD8+ T and natural killer (NK) cells and negatively regulates T and NK cell responses during hepatitis C virus (HCV) infection. However, whether LAG-3 is involved in the regulation of the antibody response remains unclear. This study aims to investigate the relationship of LAG-3 with neutralizing antibody (nAb) response during HCV infection. A total of 66 HCV-infected individuals and 36 sex- and age-matched healthy controls from a population of intravenous drug users were recruited. Circulating follicular helper T (cTfh) cells and LAG-3-expressing CD4+ T cells, type 1 regulatory T (Tr1) cells, and regulatory T (Treg) cells were characterized by flow cytometry. Serum nAb response of HCV-infected individuals was determined using pseudoparticle neutralization assays. We found that HCV infection enhanced LAG-3 expression on CD4+ T cells and exhibited regulatory T cell-like phenotype and inversely associated with the HCV nAb response. Further analysis showed that frequency of CXCR3+ cTfh cells positively correlated with nAb response, however LAG-3+ CD4+ T cells inversely associated with CXCR3+ cTfh cells. This study observed that LAG-3+ CD4+ T cells exhibit a regulatory cell phenotype and negatively associate with the HCV nAb response, implying that LAG-3 may be involved in the negative regulation of humoral immunity during HCV infection.

Development and validation of different indirect ELISAs for MERS-CoV serological testing

Since 2012, MERS-CoV has caused up to 2220 cases and 790 deaths in 27 countries with Saudi Arabia being the most affected country with ~83.1% of the cases and ~38.8% local death rate. Current serological assays such as microneutralization (MN), plaque reduction neutralization, immunofluorescence, protein microarray or pseudoparticle neutralization assays rely on handling of live MERS-CoV in high containment laboratories or need for expensive and special equipment and reagents and highly trained personnel which represent a technical hurdle for most laboratories in resource-limited MERS-CoV endemic countries. Here, we developed, compared and evaluated three different indirect ELISAs based on MERS-CoV nucleocapsid protein (N), spike (S) ectodomain (amino acids 1–1297) and S1 subunit (amino acids 1–725) and compared them with MN assay. The developed ELISAs were evaluated using large number of confirmed seropositive (79 samples) and seronegative (274 samples) MERS-CoV human serum samples. Both rS1- and rS-ELISAs maintained high sensitivity and specificity (≥90%) across a wider range of OD values compared to rN-ELISA. Moreover, rS1- and rS-based ELISAs showed better agreement and correlation with MN assay in contrast to rN-ELISA. Collectively, our data demonstrate that rS1-ELISA and rS-ELISA are more reliable than rN-ELISA and represent a suitable choice for seroepidemiological testing and surveillance in MERS-CoV endemic regions.

Circulating CXCR3 Tfh Cells Positively Correlate with Neutralizing Antibodies Responses During HCV Infection

OBJECTIVE: Circulating T follicular helper (cTfh) cells have been identified as counterparts of germinal center Tfh (GC Tfh) cells in humans and can support T-dependent B cell maturation and antibody production in vitro. However, the role of cTfh cells in neutralizing antibody responses during HCV infection remains unclear. Here, we characterized the phenotype and function of cTfh cells and demonstrated associations of cTfh cells and their subsets with neutralizing antibody responses during HCV infection. METHODS: A total of 38 HCV-infected individuals and 28 healthy controls were enrolled from a pool of injection drug users (IDUs). The frequency and function of blood Tfh cells were analyzed by flow cytometry. The titers and breadths of serum neutralizing antibodies were measured using HCV pseudo-particle neutralization assays. RESULTS: Herein, we report several key observations. First, HCV infection skewed cTfh toward CXCR3 cTfh cell differentiation. Second, the frequency of CXCR3 cTfh cells was positively correlated with HCV neutralizing antibody titers and breadths. Third, CXCR3 cTfh cells showed higher expression of Tfh-associated molecules (PD-1, ICOS, IL-21, Bcl-6) compared with CXCR3- cTfh cells from individuals with HCV infection. Coculture of cTfh cells and autologous memory B cells in vitro indicated that CXCR3 cTfh cells show a superior ability to support HCV E2-specific B cell expansion compared with CXCR3- cTfh cells from individuals with HCV infection. CONCLUSION: HCV infection skews cTfh cells toward CXCR3-biased Tfh cell differentiation, which positively correlates with the magnitude and breadth of the HCV neutralizing antibody response. It is our hope that these findings will provide insights for the rational design of a neutralizing antibody-based HCV vaccine. FUNDING STATEMENT: This study was supported in part by the Natural Science Foundation of China (grant numbers 81471959 and 81501746) and the foundation of The First People’s Hospital of Chenzhou (grant number N2018-001). DECLARATION OF INTERESTS: The authors who participated in this study declare that they have nothing to disclose regarding funding or conflicts of interest related to this manuscript. ETHICS APPROVAL STATEMENT: This study was approved by the Ethics Committee of The First People’s Hospital of Chenzhou (No. 2015002) and followed the principles established by the Declaration of Helsinki. All participants enrolled in the study provided written informed consent.

Comparison of serological assays to titrate Hantaan and Seoul hantavirus-specific antibodies

BackgroundHantaan and Seoul viruses, in the Hantavirus genus, are known to cause hemorrhagic fever with renal syndrome (HFRS). The plaque reduction neutralization test (PRNT), as conventional neutralization test for hantaviruses, is laborious and time-consuming. Alternatives to PRNT for hantaviruses are required.MethodsIn this study, the methods for Hantaan and Seoul viruses serological typing including microneutralization test (MNT), pseudoparticle neutralization test (PPNT) and immunofluorescence assay based on viral glycoproteins (IFA-GP) were developed and compared with PRNT using a panel of 74 sera including 44 convalescent sera of laboratory confirmed HFRS patients and 30 patients sera of non-hantavirus infection. Antibody titres and serotyping obtained with different methods above were analyzed by paired-t, linear correlation, McNemar χ2 and Kappa agreement tests.ResultsAntibody titres obtained with MNT50, PPNT50 and IFA-GP were significantly correlated with that obtained with PRNT50 (p < 0.001). GMT determined by PPNT50 was statistically higher than that determined by PRNT50 (p < 0.001), while GMT determined by MNT50 and IFA-GP were equal with (p > 0.05) and less than (p < 0.001) that obtained with PRNT50 respectively. Serotyping obtained with MNT50 and PRNT50, PPNT50 and PRNT50 were highly consistent (p < 0.001), whereas that obtained with IFA-GP and PRNT50 were moderately consistent (p < 0.001). There were no significant differences for serotyping between PRNT50 and MNT50, as well as PRNT50 and PPNT50 (p > 0.05). IFA-GP was less sensitive than PRNT50 and MNT50 for serotyping of hantaviruses infection (p < 0.05). However, for 79.5% (35/44) samples, serotyping determined by IFA-GP and PRNT50 were consistent.ConclusionsMNT50 and PPNT50 both can be used as simple and rapid alternatives to PRNT50, and MNT50 is more specific while PPNT50 is more sensitive than other assays for neutralizing antibody determination. So far, this work has been the most comprehensive comparison of alternatives to PRNT.

English

A cross-sectional study was conducted between October 2014 and May 2015, to determine the sero-prevalence, assess the presence of active cases through isolation and identify possible risk factors for the Middle East Respiratory Syndrome Corona Virus (MERS-CoV) in camels, in selected areas of Oromia and Afar regional states of Ethiopia. A total of 569 dromedary camel sera were collected and screened with two serological tests: Pseudo particle neutralization for screening and Micro-neutralization test for confirmation. The overall prevalence of MERS-CoV in the study area was 93% (n=529). Higher prevalence (93.9%) was recorded in female dromedary camels compared to males (91.3%) but the difference was not significant (Chi-Square=1.323 and P=0.250). Age wise prevalence was higher in adult camels (94.6%) than young ones (90.8%), however the difference was statistically insignificant (Chi-Square=3.068 and P=0.080). Similarly, higher prevalence was recorded in larger herds (93.4%) than small herds but the difference observed was not statistically significant (Chi-Square=0.220 and p=0.639). Also, no significant (Chi-Square 0.525 and P=0.469) difference was observed in prevalence between the two regions (Oromia=93.3% and Afar=93.3%). However significant difference was observed in the lower administrations of the regions (Zones, District and Kebeles) in which the highest prevalence recorded in East Shoa, Fentale district of Adis Ketema kebele (99.3%). From total of 100 swabs collected, MERS-CoV was detected in seven (1 Fentale, 4 Amibara and 2 Dubti) districts by Real-time reverse transcription polymerase chain reaction (RT-PCR). Generally, this study showed the existence of high seroprevalence of MERS-CoV among Ethiopian dromedary camels, which was also confirmed by RT-PCR. Therefore further study is required to determine its significance from both animal and public health perspectives and further research should focus on identifying similarity between MERS-CoV viral isolates in neighboring countries and clinical isolates from the Middle East and elsewhere. Key words: Dromedary camel, MERS-CoV, serum, swab, Real-time reverse transcription polymerase chain reaction (RT-PCR), Ethiopia.

A46 MERS-CoV in Arabian camels in Africa and Central Asia.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causing infections in humans is genetically indistinguishable from the virus found in Arabian camels (dromedaries) in the Middle East. Although no primary human case of MERS was reported outside the Arabian Peninsula, camel populations in Africa are known to have high prevalence of antibodies against MERS-CoV. We carried out surveillance for MERS-CoV in dromedaries in Africa and Central Asia. By MERS-CoV spike pseudoparticle neutralization assay we confirmed that camel serum samples from African countries have high prevalence of MERS-CoV antibodies. Using RT-qPCR we detected MERS-CoV positives in camel nasal swabs from all different African countries from which samples were collected. However, dromedary serum and swab samples from Kazakhstan in Central Asia were negative for MERS-CoV by these assays. Phylogenetic analysis of the spike gene revealed that MERS-CoVs from Africa formed a cluster closely related to but distinct from the viruses from the Arabian Peninsula. Results from this study suggest that MERS-CoV is actively circulating in dromedary populations in Africa and the virus in Africa is phylogenetically distinct from that in the Middle East. (Resume d'auteur)

Pseudoparticle neutralization is a reliable assay to measure immunity and cross-reactivity to H5N1 influenza viruses

The standard serological methods present limitations for the measurement of immunity against H5N1 influenza strains. The hemagglutination inhibition (HI) assay lacks sensitivity and requires standardization, while the viral micro-neutralization (MN) assay needs handling of live virus. We produced pseudoparticles expressing hemagglutinin from clades 1 or 2 H5N1 in order to measure neutralizing antibodies in human sera after prime-boost vaccination with plain or MF59-adjuvanted H5N1 clade 1 subunit vaccines. Titers measured by pseudoparticle neutralization (PPN) assay significantly correlated with those measured by HI, single radial haemolysis or MN, with a PPN titer of 1:357 corresponding to an MN titer of 1:80. Notably, results from the PPN assay, confirm that MF59-H5N1 vaccine induces potent and long-lasting neutralizing antibody responses not only against the vaccine strain, but also against several heterologous clade 2 strains. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of H5N1 vaccine immunogenicity.