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Background: Plaque Reduction Neutralization Test (PRNT) is the standard assay used for measuring neutralizing antibody responses to Herpes simplex virus type-2 (HSV-2). The PRNT is a cumbersome, time-consuming and laborious assay. The development of a faster, high throughput microneutralization assay (MNA) for HSV-2 viruses carried out in a 96-well format will allow for rapid testing of large numbers of samples for drug and vaccine development., Methods: We describe the generation of a MNA that utilizes a pair of anti-HSV human monoclonal antibodies (mAbs) for virus detection in HSV-2 infected Vero cells. Antibodies were generated by B-cell cloning from PBMC's isolated from HSV-1 negative/HSV-2 positive donors. We describe the selection and characterization of the antibodies used for virus detection by ELISA with purified, recombinant anti-HSV glycoproteins, antibody binding in infected cells, and Western Blot. We determine the anti-HSV-2 neutralizing titers of immune sera from mice by MNA and PRNT and compare these results by linear regression analysis., Results: We show that neutralization titers for HSV-2, determined by the 96-well MNA correlate with titers determined by a PRNT completed in 24-well plates in both the absence (R 2 = 0.8250) and presence (R 2 = 0.7075) of complement., Conclusions: We have successfully developed an MNA that can be used in place of the burdensome PRNT to determine anti-HSV-2 neutralizing activity in serum. This MNA has much greater throughput than the PRNT, allowing many more samples to be processed in a shorter time saving ∼90 % of the time required by the laboratory scientist to complete the task as compared to the traditional PRNT., (Copyright © 2021. Published by Elsevier B.V.)

Human cytomegalovirus (HCMV) is one of the main causative agents of congenital viral infection in neonates. HCMV infection also causes serious morbidity and mortality among organ transplant patients. Glycoprotein B (gB) is a major target for HCMV neutralizing antibodies, yet the underlying neutralization mechanisms remain largely unknown. Here we report that 3-25, a gB-specific monoclonal antibody previously isolated from a healthy HCMV-positive donor, efficiently neutralized 14 HCMV strains in both ARPE-19 cells and MRC-5 cells. The core epitope of 3-25 was mapped to a highly conserved linear epitope on antigenic domain 2 (AD-2) of gB. A 1.8 Å crystal structure of 3-25 Fab in complex with the peptide epitope revealed the molecular determinants of 3-25 binding to gB at atomic resolution. Negative-staining electron microscopy (EM) 3D reconstruction of 3-25 Fab in complex with de-glycosylated postfusion gB showed that 3-25 Fab fully occupied the gB trimer at the N-terminus with flexible binding angles. Functionally, 3-25 efficiently inhibited HCMV infection at a post-attachment step by interfering with viral membrane fusion, and restricted post-infection viral spreading in ARPE-19 cells. Interestingly, bivalency was required for HCMV neutralization by AD-2 specific antibody 3-25 but not the AD-4 specific antibody LJP538. In contrast, bivalency was not required for HCMV binding by both antibodies. Taken together, our results reveal the structural basis of gB recognition by 3-25 and demonstrate that inhibition of viral membrane fusion and a requirement of bivalency may be common for gB AD-2 specific neutralizing antibody., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interest: Tong-Ming Fu and Zhiqiang An filed a patent on 3-25 antibody.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children and older adults. Currently, no licensed vaccine is available, and therapeutic options are limited. The primary target of neutralizing antibodies to RSV is the surface fusion (F) glycoprotein. Understanding the recognition of antibodies with high neutralization potencies to RSV F antigen will provide critical insights in developing efficacious RSV antibodies and vaccines. In this study, we isolated and characterized a panel of monoclonal antibodies (mAbs) with high binding affinity to RSV prefusion F trimer and neutralization potency to RSV viruses. The mAbs were mapped to previously defined antigenic sites, and some that mapped to the same antigenic sites showed remarkable diversity in specificity, binding, and neutralization potencies. We found that the isolated site III mAbs shared highly conserved germline V-gene usage, but had different cross-reactivities to human metapneumovirus (hMPV), possibly due to the distinct modes/angles of interaction with RSV and hMPV F proteins. Furthermore, we identified a subset of potent RSV/hMPV cross-neutralizing mAbs that target antigenic site IV and the recently defined antigenic site V, while the majority of the mAbs targeting these two sites only neutralize RSV. Additionally, the isolated mAbs targeting site Ø were mono-specific for RSV and showed a wide range of neutralizing potencies on different RSV subtypes. Our data exemplify the diversity of anti-RSV mAbs and provide new insights into the immune recognition of respiratory viruses in the Pneumoviridae family.

Respiratory syncytial virus (RSV) infection is the leading cause of hospitalization and infant mortality under six months of age worldwide; therefore, the prevention of RSV infection in all infants represents a significant unmet medical need. Here we report the isolation of a potent and broadly neutralizing RSV monoclonal antibody derived from a human memory B-cell. This antibody, RB1, is equipotent on RSV A and B subtypes, potently neutralizes a diverse panel of clinical isolates in vitro and demonstrates in vivo protection. It binds to a highly conserved epitope in antigenic site IV of the RSV fusion glycoprotein. RB1 is the parental antibody to MK-1654 which is currently in clinical development for the prevention of RSV infection in infants.

Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection, and developing a prophylactic vaccine is of high priority to public health. We recently reported a replication-defective human cytomegalovirus with restored pentameric complex glycoprotein H (gH)/gL/pUL128-131 for prevention of congenital HCMV infection. While the quantity of vaccine-induced antibody responses can be measured in a viral neutralization assay, assessing the quality of such responses, including the ability of vaccine-induced antibodies to cross-neutralize the field strains of HCMV, remains a challenge. In this study, with a panel of neutralizing antibodies from three healthy human donors with natural HCMV infection or a vaccinated animal, we mapped eight sites on the dominant virus-neutralizing antigen-the pentameric complex of glycoprotein H (gH), gL, and pUL128, pUL130, and pUL131. By evaluating the site-specific antibodies in vaccine immune sera, we demonstrated that vaccination elicited functional antiviral antibodies to multiple neutralizing sites in rhesus macaques, with quality attributes comparable to those of CMV hyperimmune globulin. Furthermore, these immune sera showed antiviral activities against a panel of genetically distinct HCMV clinical isolates. These results highlighted the importance of understanding the quality of vaccine-induced antibody responses, which includes not only the neutralizing potency in key cell types but also the ability to protect against the genetically diverse field strains. IMPORTANCE HCMV is the leading cause of congenital viral infection, and development of a preventive vaccine is a high public health priority. To understand the strain coverage of vaccine-induced immune responses in comparison with natural immunity, we used a panel of broadly neutralizing antibodies to identify the immunogenic sites of a dominant viral antigen-the pentameric complex. We further demonstrated that following vaccination of a replication-defective virus with the restored pentameric complex, rhesus macaques can develop broadly neutralizing antibodies targeting multiple immunogenic sites of the pentameric complex. Such analyses of site-specific antibody responses are imperative to our assessment of the quality of vaccine-induced immunity in clinical studies., (Copyright © 2017 Ha et al.)

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F) glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis® (palivizumab) is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs) against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction.

Monitoring antigen-specific memory B cells and the antibodies they encode is important for understanding the specificity, breadth and duration of immune response to an infection or vaccination. The antibodies isolated could further help design vaccine antigens for raising relevant protective immune responses. However, developing assays to measure and isolate antigen-specific memory B cells is technically challenging due to the low frequencies of these cells that exist in the circulating blood. Here, we describe a flow cytometry method to identify and isolate dengue envelope-specific memory B cells using a labeled dengue envelope protein. We enumerated dengue-envelope specific memory B cells from a cohort of dengue seropositive donors using this direct flow cytometry assay. A more established and conventional assay, the cultured B ELISPOT, was used as a benchmark comparator. Furthermore, we were able to confirm the single-sorted memory B-cell specificity by culturing B cells and differentiating them into plasma cells using cell lines expressing CD40L. The culture supernatants were assayed for antigen binding and the ability of the antibodies to neutralize the cognate dengue virus. Moreover, we successfully isolated the heavy and light Ig sequences and expressed them as full-length recombinant antibodies to reproduce the activity seen in culture supernatants. Mapping of these antibodies revealed a novel epitope for dengue 2 virus serotype. In conclusion, we established a reproducible methodology to enumerate antigen-specific memory B cells and assay their encoded antibodies for functional characterization.

Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.

Human cytomegalovirus (HCMV) can cause serious morbidity/mortality in transplant patients, and congenital HCMV infection can lead to birth defects. Developing an effective HCMV vaccine is a high medical priority. One of the challenges to the efforts has been our limited understanding of the viral antigens important for protective antibodies. Receptor-mediated viral entry to endothelial/epithelial cells requires a glycoprotein H (gH) complex comprising five viral proteins (gH, gL, UL128, UL130, and UL131). This gH complex is notably missing from HCMV laboratory strains as well as HCMV vaccines previously evaluated in the clinic. To support a unique vaccine concept based on the pentameric gH complex, we established a panel of 45 monoclonal antibodies (mAbs) from a rabbit immunized with an experimental vaccine virus in which the expression of the pentameric gH complex was restored. Over one-half (25 of 45) of the mAbs have neutralizing activity. Interestingly, affinity for an antibody to bind virions was not correlated with its ability to neutralize the virus. Genetic analysis of the 45 mAbs based on their heavy- and light-chain sequences identified at least 26 B-cell linage groups characterized by distinct binding or neutralizing properties. Moreover, neutralizing antibodies possessed longer complementarity-determining region 3 for both heavy and light chains than those with no neutralizing activity. Importantly, potent neutralizing mAbs reacted to the pentameric gH complex but not to gB. Thus, the pentameric gH complex is the primary target for antiviral antibodies by vaccination.

Naturally acquired immunity significantly reduces the risk of congenital cytomegalovirus (CMV) infection in future pregnancies. An immune response comparable to that of natural infection has been used as a benchmark for CMV vaccine efficacy; however, the magnitude and persistence of the neutralizing antibody responses in naturally infected women are not completely understood. In this study, we quantitatively analyzed a panel of 375 female CMV convalescent sera ranging in age from 18 to 84 years, for its ability to block virus entry into epithelial cells and fibroblasts, as well as its binding potential to CMV particles. The geometric mean titer of the sera in this panel to neutralize 50% of the virus entry into epithelial cells was 7491, compared to 802 for entry into fibroblasts. The epithelial neutralizing titers were statistically indistinguishable among different age groups, and conformed to a normal distribution. There was a weak correlation between the levels of neutralization and the binding activities to viral particles. Our data confirmed that natural CMV infection in healthy women induces potent neutralizing antibodies against infection of both fibroblasts and epithelial cells. The serum neutralizing activities were maintained at high levels throughout the child bearing age. The corresponding titers may serve as a biomarker for CMV vaccine efficacy., (Copyright © 2011 Elsevier Ltd. All rights reserved.)