Publications by authors named "Surender Khurana"

73 Publications

Longitudinal antibody repertoire in "mild" versus "severe" COVID-19 patients reveals immune markers associated with disease severity and resolution.

Sci Adv 2021 03 5;7(10). Epub 2021 Mar 5.

Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD 20871, USA.

Limited knowledge exists on immune markers associated with disease severity or recovery in patients with coronavirus disease 2019 (COVID-19). Here, we elucidated longitudinal evolution of SARS-CoV-2 antibody repertoire in patients with acute COVID-19. Differential kinetics was observed for immunoglobulin M (IgM)/IgG/IgA epitope diversity, antibody binding, and affinity maturation in "severe" versus "mild" COVID-19 patients. IgG profile demonstrated immunodominant antigenic sequences encompassing fusion peptide and receptor binding domain (RBD) in patients with mild COVID-19 who recovered early compared with "fatal" COVID-19 patients. In patients with severe COVID-19, high-titer IgA were observed, primarily against RBD, especially in patients who succumbed to SARS-CoV-2 infection. The patients with mild COVID-19 showed marked increase in antibody affinity maturation to prefusion SARS-CoV-2 spike that associated with faster recovery from COVID-19. This study revealed antibody markers associated with disease severity and resolution of clinical disease that could inform development and evaluation of effective immune-based countermeasures against COVID-19.
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http://dx.doi.org/10.1126/sciadv.abf2467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935365PMC
March 2021

Antibody affinity maturation and plasma IgA associate with clinical outcome in hospitalized COVID-19 patients.

Nat Commun 2021 02 22;12(1):1221. Epub 2021 Feb 22.

Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, USA.

Hospitalized COVID-19 patients often present with a large spectrum of clinical symptoms. There is a critical need to better understand the immune responses to SARS-CoV-2 that lead to either resolution or exacerbation of the clinical disease. Here, we examine longitudinal plasma samples from hospitalized COVID-19 patients with differential clinical outcome. We perform immune-repertoire analysis including cytokine, hACE2-receptor inhibition, neutralization titers, antibody epitope repertoire, antibody kinetics, antibody isotype and antibody affinity maturation against the SARS-CoV-2 prefusion spike protein. Fatal cases demonstrate high plasma levels of IL-6, IL-8, TNFα, and MCP-1, and sustained high percentage of IgA-binding antibodies to prefusion spike compared with non-ICU survivors. Disease resolution in non-ICU and ICU patients associates with antibody binding to the receptor binding motif and fusion peptide, and antibody affinity maturation to SARS-CoV-2 prefusion spike protein. Here, we provide insight into the immune parameters associated with clinical disease severity and disease-resolution outcome in hospitalized patients that could inform development of vaccine/therapeutics against COVID-19.
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http://dx.doi.org/10.1038/s41467-021-21463-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900119PMC
February 2021

Nonhuman primates exposed to Zika virus in utero are not protected against reinfection at 1 year postpartum.

Sci Transl Med 2020 10;12(567)

Emerging Pathogens Section, Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA.

There is limited information about the impact of Zika virus (ZIKV) exposure in utero on the anti-ZIKV immune responses of offspring. We infected six rhesus macaque dams with ZIKV early or late in pregnancy and studied four of their offspring over the course of a year postpartum. Despite evidence of ZIKV exposure in utero, we observed no structural brain abnormalities in the offspring. We detected infant-derived ZIKV-specific immunoglobulin A antibody responses and T cell memory responses during the first year postpartum in the two offspring born to dams infected with ZIKV early in pregnancy. Critically, although the infants had acquired some immunological memory of ZIKV, it was not sufficient to protect them against reinfection with ZIKV at 1 year postpartum. The four offspring reexposed to ZIKV at 1 year postpartum all survived but exhibited acute viremia and viral tropism to lymphoid tissues; three of four reexposed offspring exhibited spinal cord pathology. These data suggest that macaque infants born to dams infected with ZIKV during pregnancy remain susceptible to postnatal infection and consequent neuropathology.
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http://dx.doi.org/10.1126/scitranslmed.aaz4997DOI Listing
October 2020

Autoreactivity of Broadly Neutralizing Influenza Human Antibodies to Human Tissues and Human Proteins.

Viruses 2020 10 8;12(10). Epub 2020 Oct 8.

Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD 20993, USA.

Broadly neutralizing monoclonal antibodies (bNAbs) against conserved domains in the influenza hemagglutinin are in clinical trials. Several next generation influenza vaccines designed to elicit such bNAbs are also in clinical development. One of the common features of the isolated bNAbs is the use of restricted IgV repertoire. More than 80% of stem-targeting bNAbs express IgV1-69, which may indicate genetic constraints on the evolution of such antibodies. In the current study, we evaluated a panel of influenza virus bNAbs in comparison with HIV-1 MAb 4E10 and anti-RSV MAb Palivizumab (approved for human use) for autoreactivity using 30 normal human tissues microarray and human protein (>9000) arrays. We found that several human bNAbs (CR6261, CR9114, and F2603) reacted with human tissues, especially with pituitary gland tissue. Importantly, protein array analysis identified high-affinity interaction of CR6261 with the autoantigen "Enhancer of mRNA decapping 3 homolog" (EDC3), which was not previously described. Moreover, EDC3 competed with hemagglutinin for binding to bNAb CR6261. These autoreactivity findings underscores the need for careful evaluation of such bNAbs for therapeutics and stem-based vaccines against influenza virus.
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http://dx.doi.org/10.3390/v12101140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600923PMC
October 2020

Bromelain Inhibits SARS-CoV-2 Infection in VeroE6 Cells.

bioRxiv 2020 Sep 16. Epub 2020 Sep 16.

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska, USA 68198.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The initial interaction between Transmembrane Serine Protease 2 (TMPRSS2) primed SARS-CoV-2 spike (S) protein and host cell receptor angiotensin-converting enzyme 2 (ACE-2) is a pre-requisite step for this novel coronavirus pathogenesis. Here, we expressed a GFP-tagged SARS-CoV-2 S-Ectodomain in Tni insect cells. That contained sialic acid-enriched N- and O-glycans. Surface resonance plasmon (SPR) and Luminex assay showed that the purified S-Ectodomain binding to human ACE-2 and immunoreactivity with COVID-19 positive samples. We demonstrate that bromelain (isolated from pineapple stem and used as a dietary supplement) treatment diminishes the expression of ACE-2 and TMPRSS2 in VeroE6 cells and dramatically lowers the expression of S-Ectodomain. Importantly, bromelain treatment reduced the interaction between S-Ectodomain and VeroE6 cells. Most importantly, bromelain treatment significantly diminished the SARS-CoV-2 infection in VeroE6 cells. Altogether, our results suggest that bromelain or bromelain rich pineapple stem may be used as an antiviral against COVID-19.

Highlights: Bromelain inhibits / cleaves the expression of ACE-2 and TMPRSS2Bromelain cleaves / degrades SARS-CoV-2 spike proteinBromelain inhibits S-Ectodomain binding and SARS-CoV-2 infection.
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http://dx.doi.org/10.1101/2020.09.16.297366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523097PMC
September 2020

Antibody signature induced by SARS-CoV-2 spike protein immunogens in rabbits.

Sci Transl Med 2020 07 8;12(550). Epub 2020 Jun 8.

Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD 20871, USA.

Multiple vaccine candidates against SARS-CoV-2 based on viral spike protein are under development. However, there is limited information on the quality of antibody responses generated with these vaccine modalities. To better understand antibody responses induced by spike protein-based vaccines, we performed a qualitative study by immunizing rabbits with various SARS-CoV-2 spike protein antigens: S ectodomain (S1+S2; amino acids 16 to 1213), which lacks the cytoplasmic and transmembrane domains (CT-TM), the S1 domain (amino acids 16 to 685), the receptor binding domain (RBD) (amino acids 319 to 541), and the S2 domain (amino acids 686 to 1213, lacking the RBD, as control). Resulting antibody quality and function were analyzed by enzyme-linked immunosorbent assay (ELISA), RBD competition assay, surface plasmon resonance (SPR) against different spike proteins in native conformation, and neutralization assays. All three antigens (S1+S2 ectodomain, S1 domain, and RBD), but not S2, generated strong neutralizing antibodies against SARS-CoV-2. Vaccination-induced antibody repertoire was analyzed by SARS-CoV-2 spike genome fragment phage display libraries (SARS-CoV-2 GFPDL), which identified immunodominant epitopes in the S1, S1-RBD, and S2 domains. Furthermore, these analyses demonstrated that the RBD immunogen elicited a higher antibody titer with five-fold higher affinity antibodies to native spike antigens compared with other spike antigens, and antibody affinity correlated strongly with neutralization titers. These findings may help guide rational vaccine design and facilitate development and evaluation of effective therapeutics and vaccines against COVID-19 disease.
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http://dx.doi.org/10.1126/scitranslmed.abc3539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7286538PMC
July 2020

Human Antibody Repertoire following Ebola Virus Infection and Vaccination.

iScience 2020 Mar 19;23(3):100920. Epub 2020 Feb 19.

Division of Viral Products, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), 10903 New Hampshire Avenue, Silver Spring, MD 20993, USA. Electronic address:

Limited knowledge exists on the quality of polyclonal antibody response generated following Ebola virus (EBOV) infection compared with vaccination. Polyclonal antibody repertoire in plasma following EBOV infection in survivors was compared with ChAd3-MVA prime-boost human vaccination. Higher antibody binding and affinity to GP was observed in survivors compared with vaccinated plasma that correlated with EBOV neutralization. Surprisingly, a predominant IgM response was generated after prime-boost vaccination, whereas survivors demonstrated IgG-dominant antibody response. EBOV infection induced more diverse antibody epitope repertoire compared with vaccination. A strong binding to antigenic sites in the fusion peptide and another in the highly conserved GP2-HR2 domain was preferentially recognized by EBOV survivors than vaccinated individuals that correlated strongly with EBOV neutralization titers. These findings will help development and evaluation of effective Ebola countermeasures including therapeutics and vaccines.
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http://dx.doi.org/10.1016/j.isci.2020.100920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058406PMC
March 2020

Longitudinal Human Antibody Repertoire against Complete Viral Proteome from Ebola Virus Survivor Reveals Protective Sites for Vaccine Design.

Cell Host Microbe 2020 02;27(2):262-276.e4

Critical Care Medicine Department, National Institutes of Health Clinical Center, Bethesda, MD 20892, USA; Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Evolution of antibody repertoire against the Ebola virus (EBOV) proteome was characterized in an acutely infected patient receiving supportive care alone to elucidate virus-host interactions over time. Differential kinetics are observed for IgM-IgG-IgA epitope diversity, antibody binding, and affinity maturation to EBOV proteins. During acute illness, antibodies predominate to VP40 and glycoprotein (GP). At day 13 of clinical illness, a marked increase in antibody titers to most EBOV proteins and affinity maturation to GP is associated with rapid decline in viral replication and illness severity. At one year, despite undetectable virus, a diverse IgM repertoire against VP40 and GP epitopes is observed suggesting occult viral persistence. Rabbit immunization experiments identify key immunodominant sites of GP, while challenge studies in mice found these epitopes induce EBOV-neutralizing antibodies and protect against lethal EBOV challenge. This study reveals markers of viral persistence and provides promising approaches for development and evaluation of vaccines and therapeutics.
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http://dx.doi.org/10.1016/j.chom.2020.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7071344PMC
February 2020

Antigenic Fingerprinting of Respiratory Syncytial Virus (RSV)-A-Infected Hematopoietic Cell Transplant Recipients Reveals Importance of Mucosal Anti-RSV G Antibodies in Control of RSV Infection in Humans.

J Infect Dis 2020 02;221(4):636-646

Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.

Background: Respiratory syncytial virus (RSV) infection causes significant morbidity in hematopoietic cell transplant (HCT) recipients. However, antibody responses that correlate with recovery from RSV disease are not fully understood.

Methods: In this study, antibody repertoire in paired serum and nasal wash samples from acutely RSV-A-infected HCT recipients who recovered early (<14 days of RSV shedding) were compared with late-recovered patients (≥14 days of shedding) using gene fragment phage display libraries and surface plasmon resonance.

Results: Anti-F serum responses were similar between these 2 groups for antibody repertoires, neutralization titers, anti-F binding antibodies (prefusion and postfusion proteins), antibody avidity, and binding to specific antigenic sites. In contrast, nasal washes from early-recovered individuals demonstrated higher binding to F peptide containing p27. While the serum RSV G antibody repertoires in the 2 groups were similar, the strongest difference between early-recovered and late-recovered patients was observed in the titers of nasal wash antibodies, especially binding to the central conserved domain. Most importantly, a significantly higher antibody affinity to RSV G was observed in nasal washes from early-recovered individuals compared with late-recovered HCT recipients.

Conclusions: These findings highlight the importance of mucosal antibodies in resolution of RSV-A infection in the upper respiratory tract.
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http://dx.doi.org/10.1093/infdis/jiz608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530544PMC
February 2020

Anti-influenza hyperimmune intravenous immunoglobulin for adults with influenza A or B infection (FLU-IVIG): a double-blind, randomised, placebo-controlled trial.

Lancet Respir Med 2019 11 30;7(11):951-963. Epub 2019 Sep 30.

Division of Biostatistics, School of Public Health, University of Minnesota, Minneapolis, MN, USA.

Background: Since the 1918 influenza pandemic, non-randomised studies and small clinical trials have suggested that convalescent plasma or anti-influenza hyperimmune intravenous immunoglobulin (hIVIG) might have clinical benefit for patients with influenza infection, but definitive data do not exist. We aimed to evaluate the safety and efficacy of hIVIG in a randomised controlled trial.

Methods: This randomised, double-blind, placebo-controlled trial was planned for 45 hospitals in Argentina, Australia, Denmark, Greece, Mexico, Spain, Thailand, UK, and the USA over five influenza seasons from 2013-14 to 2017-18. Adults (≥18 years of age) were admitted for hospital treatment with laboratory-confirmed influenza A or B infection and were randomly assigned (1:1) to receive standard care plus either a single 500-mL infusion of high-titre hIVIG (0·25 g/kg bodyweight, 24·75 g maximum; hIVIG group) or saline placebo (placebo group). Eligible patients had a National Early Warning score of 2 points or greater at the time of screening and their symptoms began no more than 7 days before randomisation. Pregnant and breastfeeding women were excluded, as well as any patients for whom the treatment would present a health risk. Separate randomisation schedules were generated for each participating clinical site using permuted block randomisation. Treatment assignments were obtained using a web-based application by the site pharmacist who then masked the solution for infusion. Patients and investigators were masked to study treatment. The primary endpoint was a six-category ordinal outcome of clinical status at day 7, ranging in severity from death to resumption of normal activities after discharge. The choice of day 7 was based on haemagglutination inhibition titres from a pilot study. It was analysed with a proportional odds model, using all six categories to estimate a common odds ratio (OR). An OR greater than 1 indicated that, for a given category, patients in the hIVIG group were more likely to be in a better category than those in the placebo group. Prespecified primary analyses for safety and efficacy were based on patients who received an infusion and for whom eligibility could be confirmed. This trial is registered with ClinicalTrials.gov, NCT02287467.

Findings: 313 patients were enrolled in 34 sites between Dec 11, 2014, and May 28, 2018. We also used data from 16 patients enrolled at seven of the 34 sites during the pilot study between Jan 15, 2014, and April 10, 2014. 168 patients were randomly assigned to the hIVIG group and 161 to the placebo group. 21 patients were excluded (12 from the hIVIG group and 9 from the placebo group) because they did not receive an infusion or their eligibility could not be confirmed. Thus, 308 were included in the primary analysis. hIVIG treatment produced a robust rise in haemagglutination inhibition titres against influenza A and smaller rises in influenza B titres. Based on the proportional odds model, the OR on day 7 was 1·25 (95% CI 0·79-1·97; p=0·33). In subgroup analyses for the primary outcome, the OR in patients with influenza A was 0·94 (0·55-1·59) and was 3·19 (1·21-8·42) for those with influenza B (interaction p=0·023). Through 28 days of follow-up, 47 (30%) of 156 patients in the hIVIG group and in 45 (30%) of 152 patients in the placebo group had the composite safety outcome of death, a serious adverse event, or a grade 3 or 4 adverse event (hazard ratio [HR] 1·06, 95% CI 0·70-1·60; p=0·79). Six (4%) patients in the hIVIG group and five (3%) in the placebo group died, but these deaths were not necessarily related to treatment.

Interpretation: When administered alongside standard care (most commonly oseltamivir), hIVIG was not superior to placebo for adults hospitalised with influenza infection. By contrast with our prespecified subgroup hypothesis that hIVIG would result in more favourable responses in patients with influenza A than B, we found the opposite effect. The clinical benefit of hIVIG for patients with influenza B is supported by antibody affinity analyses, but confirmation is warranted.

Funding: NIAID and NIH. Partial support was provided by the Medical Research Council (MRC_UU_12023/23) and the Danish National Research Foundation.
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http://dx.doi.org/10.1016/S2213-2600(19)30253-XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868512PMC
November 2019

Antibiotics-Driven Gut Microbiome Perturbation Alters Immunity to Vaccines in Humans.

Cell 2019 09;178(6):1313-1328.e13

Institute for Immunity, Transplantation and Infection, School of Medicine, Stanford University, Stanford, CA 94305, USA; Department of Pathology, Stanford University School of Medicine, Stanford University, Stanford, CA 94305, USA; Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford University, Stanford, CA 94305, USA. Electronic address:

Emerging evidence indicates a central role for the microbiome in immunity. However, causal evidence in humans is sparse. Here, we administered broad-spectrum antibiotics to healthy adults prior and subsequent to seasonal influenza vaccination. Despite a 10,000-fold reduction in gut bacterial load and long-lasting diminution in bacterial diversity, antibody responses were not significantly affected. However, in a second trial of subjects with low pre-existing antibody titers, there was significant impairment in H1N1-specific neutralization and binding IgG1 and IgA responses. In addition, in both studies antibiotics treatment resulted in (1) enhanced inflammatory signatures (including AP-1/NR4A expression), observed previously in the elderly, and increased dendritic cell activation; (2) divergent metabolic trajectories, with a 1,000-fold reduction in serum secondary bile acids, which was highly correlated with AP-1/NR4A signaling and inflammasome activation. Multi-omics integration revealed significant associations between bacterial species and metabolic phenotypes, highlighting a key role for the microbiome in modulating human immunity.
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http://dx.doi.org/10.1016/j.cell.2019.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6750738PMC
September 2019

Repeat vaccination reduces antibody affinity maturation across different influenza vaccine platforms in humans.

Nat Commun 2019 07 26;10(1):3338. Epub 2019 Jul 26.

Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, 20993, USA.

Several vaccines are approved in the United States for seasonal influenza vaccination every year. Here we compare the impact of repeat influenza vaccination on hemagglutination inhibition (HI) titers, antibody binding and affinity maturation to individual hemagglutinin (HA) domains, HA1 and HA2, across vaccine platforms. Fold change in HI and antibody binding to HA1 trends higher for H1N1pdm09 and H3N2 but not against B strains in groups vaccinated with FluBlok compared with FluCelvax and Fluzone. Antibody-affinity maturation occurs against HA1 domain of H1N1pdm09, H3N2 and B following vaccination with all vaccine platforms, but not against H1N1pdm09-HA2. Importantly, prior year vaccination of subjects receiving repeat vaccinations demonstrated reduced antibody-affinity maturation to HA1 of all three influenza virus strains irrespective of the vaccine platform. This study identifies an important impact of repeat vaccination on antibody-affinity maturation following vaccination, which may contribute to lower vaccine effectiveness of seasonal influenza vaccines in humans.
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http://dx.doi.org/10.1038/s41467-019-11296-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6659679PMC
July 2019

Antibody-dependent enhancement of influenza disease promoted by increase in hemagglutinin stem flexibility and virus fusion kinetics.

Proc Natl Acad Sci U S A 2019 07 11;116(30):15194-15199. Epub 2019 Jul 11.

Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993;

Several next-generation (universal) influenza vaccines and broadly neutralizing antibodies (bNAbs) are in clinical development. Some of these mediate inhibitions of virus replication at the postentry stage or use Fc-dependent mechanisms. Nonneutralizing antibodies have the potential to mediate enhancement of viral infection or disease. In the current study, two monoclonal antibodies (MAbs) 72/8 and 69/1, enhanced respiratory disease (ERD) in mice following H3N2 virus challenge by demonstrating increased lung pathology and changes in lung cytokine/chemokine levels. MAb 78/2 caused changes in the lung viral loads in a dose-dependent manner. Both MAbs increased HA sensitivity to trypsin cleavage at a higher pH range, suggesting MAb-induced conformational changes. pHrodo-labeled virus particles' entry and residence time in the endocytic compartment were tracked during infection of Madin-Darby canine kidney (MDCK) cells. Both MAbs reduced H3N2 virus residence time in the endocytic pathway, suggesting faster virus fusion kinetics. Structurally, 78/2 and 69/1 Fabs bound the globular head or base of the head domain of influenza hemagglutinin (HA), respectively, and induced destabilization of the HA stem domain. Together, this study describes Mab-induced destabilization of the influenza HA stem domain, faster kinetics of influenza virus fusion, and ERD in vivo. The in vivo animal model and in vitro assays described could augment preclinical safety evaluation of antibodies and next-generation influenza vaccines that generate antibodies which do not block influenza virus-receptor interaction.
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http://dx.doi.org/10.1073/pnas.1821317116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6660725PMC
July 2019

Differential human antibody repertoires following Zika infection and the implications for serodiagnostics and disease outcome.

Nat Commun 2019 04 26;10(1):1943. Epub 2019 Apr 26.

Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, 20993, USA.

Zika virus (ZIKV) outbreak in Americas led to extensive efforts to develop vaccines and ZIKV-specific diagnostics. In the current study, we use whole genome phage display library spanning the entire ZIKV genome (ZIKV-GFPDL) for in-depth immune profiling of IgG and IgM antibody repertoires in serum and urine longitudinal samples from individuals acutely infected with ZIKV. We observe a very diverse IgM immune repertoire encompassing the entire ZIKV polyprotein on day 0 in both serum and urine. ZIKV-specific IgG antibodies increase 10-fold between day 0 and day 7 in serum, but not in urine; these are highly focused on prM/E, NS1 and NS2B. Differential antibody affinity maturation is observed against ZIKV structural E protein compared with nonstructural protein NS1. Serum antibody affinity to ZIKV-E protein inversely correlates with ZIKV disease symptoms. Our study provides insight into unlinked evolution of immune response to ZIKV infection and identified unique targets for ZIKV serodiagnostics.
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http://dx.doi.org/10.1038/s41467-019-09914-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486612PMC
April 2019

Prolonged evolution of the memory B cell response induced by a replicating adenovirus-influenza H5 vaccine.

Sci Immunol 2019 04;4(34)

HIV-Specific Immunity Section of the Laboratory of Immunoregulation, National Institutes of Health (NIH), Bethesda, MD 20892, USA.

Induction of an antibody response capable of recognizing highly diverse strains is a major obstacle to the development of vaccines for viruses such as HIV and influenza. Here, we report the dynamics of B cell expansion and evolution at the single-cell level after vaccination with a replication-competent adenovirus type 4 recombinant virus expressing influenza H5 hemagglutinin. Fluorescent H1 or H5 probes were used to quantitate and isolate peripheral blood B cells and their antigen receptors. We observed increases in H5-specific antibody somatic hypermutation and potency for several months beyond the period of active viral replication that was not detectable at the serum level. Individual broad and potent antibodies could be isolated, including one stem-specific antibody that is part of a new multidonor class. These results demonstrate prolonged evolution of the B cell response for months after vaccination and should be considered in efforts to evaluate or boost vaccine-induced immunity.
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http://dx.doi.org/10.1126/sciimmunol.aau2710DOI Listing
April 2019

Broad Hemagglutinin-Specific Memory B Cell Expansion by Seasonal Influenza Virus Infection Reflects Early-Life Imprinting and Adaptation to the Infecting Virus.

J Virol 2019 04 3;93(8). Epub 2019 Apr 3.

David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, USA

Memory B cells (MBCs) are key determinants of the B cell response to influenza virus infection and vaccination, but the effect of different forms of influenza antigen exposure on MBC populations has received little attention. We analyzed peripheral blood mononuclear cells and plasma collected following human H3N2 influenza infection to investigate the relationship between hemagglutinin-specific antibody production and changes in the size and character of hemagglutinin-reactive MBC populations. Infection produced increased concentrations of plasma IgG reactive to the H3 head of the infecting virus, to the conserved stalk, and to a broad chronological range of H3s consistent with original antigenic sin responses. H3-reactive IgG MBC expansion after infection included reactivity to head and stalk domains. Notably, expansion of H3 head-reactive MBC populations was particularly broad and reflected original antigenic sin patterns of IgG production. Findings also suggest that early-life H3N2 infection "imprints" for strong H3 stalk-specific MBC expansion. Despite the breadth of MBC expansion, the MBC response included an increase in affinity for the H3 head of the infecting virus. Overall, our findings indicate that H3-reactive MBC expansion following H3N2 infection is consistent with maintenance of response patterns established early in life, but nevertheless includes MBC adaptation to the infecting virus. Rapid and vigorous virus-specific antibody responses to influenza virus infection and vaccination result from activation of preexisting virus-specific memory B cells (MBCs). Understanding the effects of different forms of influenza virus exposure on MBC populations is therefore an important guide to the development of effective immunization strategies. We demonstrate that exposure to the influenza hemagglutinin via natural infection enhances broad protection through expansion of hemagglutinin-reactive MBC populations that recognize head and stalk regions of the molecule. Notably, we show that hemagglutinin-reactive MBC expansion reflects imprinting by early-life infection and that this might apply to stalk-reactive, as well as to head-reactive, MBCs. Our findings provide experimental support for the role of MBCs in maintaining imprinting effects and suggest a mechanism by which imprinting might confer heterosubtypic protection against avian influenza viruses. It will be important to compare our findings to the situation after influenza vaccination.
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http://dx.doi.org/10.1128/JVI.00169-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450111PMC
April 2019

AS03-adjuvanted H5N1 vaccine promotes antibody diversity and affinity maturation, NAI titers, cross-clade H5N1 neutralization, but not H1N1 cross-subtype neutralization.

NPJ Vaccines 2018 1;3:40. Epub 2018 Oct 1.

1Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD 20993 USA.

Immune responses to inactivated vaccines against avian influenza are poor due in part to lack of immune memory. Adjuvants significantly increased virus neutralizing titers. We performed comprehensive analyses of polyclonal antibody responses following FDA-approved adjuvanted H5N1-A/Indonesia vaccine, administered in presence or absence of AS03. Using Whole Genome Fragment Phage Display Libraries, we observed that AS03 induced antibody epitope diversity to viral hemagglutinin (HA) and neuraminidase compared with unadjuvanted vaccine. Furthermore, AS03 promoted significant antibody affinity maturation to properly folded H5-HA1 (but not to HA2) domain, which correlated with neutralization titers against both vaccine and heterologous H5N1 strains. However, no increase in heterosubtypic cross-neutralization of Group1-H1N1 seasonal strains was observed. AS03-H5N1 vaccine also induced higher neuraminidase inhibition antibody titers. This study provides insight into the differential impacts of AS03 adjuvant on H5N1 vaccine-induced antibody responses that may help optimize vaccine platforms for future vaccines with improved protection against seasonal and pandemic influenza strains.
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http://dx.doi.org/10.1038/s41541-018-0076-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167326PMC
October 2018

Protective antigenic sites in respiratory syncytial virus G attachment protein outside the central conserved and cysteine noose domains.

PLoS Pathog 2018 08 24;14(8):e1007262. Epub 2018 Aug 24.

Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, United States of America.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract disease in infants. Previously, we elucidated the antibody repertoire following primary RSV infection in infants. Whole genome-fragment phage display libraries (GFPDL) expressing linear and conformational epitopes from RSV bound 100-fold more phages within attachment protein (G) following primary RSV infection. The G-reactive epitopes spanned the N- and C-termini of G ectodomain, in addition to the central conserved domain (CCD). In the current study, we examined the contribution of antigenic regions of G outside of the CCD to RSV-specific immunity. We evaluated the immunogenicity, neutralization and protective efficacy of all RSV-G antigenic sites identified following primary RSV infection using recombinant E. coli expressed G ectodomain (REG), CCD-deleted G ectodomain (REG ΔCCD), N- and C-terminal G subdomains, and antigenic site peptides. The REG ΔCCD, N- and C-terminal subdomains and peptides generated antibody titers in rabbits and mice that bound fully glycosylated Recombinant Mammalian expressed G ectodomain (RMG) and intact RSV virion particles but minimal in vitro neutralization titers compared with the intact G ectodomain. Vaccinated mice were challenged intranasally with RSV-A2 Line 19F. Viral replication in nasal cavity and lungs was significantly reduced in vaccinated animals compared to unimmunized controls. Control of viral loads post-RSV challenge correlated with serum antibody binding to the virus particles. In addition, very low Th2/Th1 cytokine ratios were found in the lungs of REG ΔCCD vaccinated mice after challenge. These data demonstrate the presence of multiple protective sites in RSV G protein outside of the CCD that could contribute to the development of a bacterially produced unglycosylated G protein as safe and protective vaccine against RSV disease.
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http://dx.doi.org/10.1371/journal.ppat.1007262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126872PMC
August 2018

Fully Human Immunoglobulin G From Transchromosomic Bovines Treats Nonhuman Primates Infected With Ebola Virus Makona Isolate.

J Infect Dis 2018 11;218(suppl_5):S636-S648

Integrated Research Facility at Fort Detrick, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland.

Transchromosomic bovines (Tc-bovines) adaptively produce fully human polyclonal immunoglobulin (Ig)G antibodies after exposure to immunogenic antigen(s). The National Interagency Confederation for Biological Research and collaborators rapidly produced and then evaluated anti-Ebola virus IgG immunoglobulins (collectively termed SAB-139) purified from Tc-bovine plasma after sequential hyperimmunization with an Ebola virus Makona isolate glycoprotein nanoparticle vaccine. SAB-139 was characterized by several in vitro production, research, and clinical level assays using wild-type Makona-C05 or recombinant virus/antigens from different Ebola virus variants. SAB-139 potently activates natural killer cells, monocytes, and peripheral blood mononuclear cells and has high-binding avidity demonstrated by surface plasmon resonance. SAB-139 has similar concentrations of galactose-α-1,3-galactose carbohydrates compared with human-derived intravenous Ig, and the IgG1 subclass antibody is predominant. All rhesus macaques infected with Ebola virus/H.sapiens-tc/GIN/2014/Makona-C05 and treated with sufficient SAB-139 at 1 day (n = 6) or 3 days (n = 6) postinfection survived versus 0% of controls. This study demonstrates that Tc-bovines can produce pathogen-specific human Ig to prevent and/or treat patients when an emerging infectious disease either threatens to or becomes an epidemic.
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http://dx.doi.org/10.1093/infdis/jiy377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249570PMC
November 2018

Antibody Repertoire of Human Polyclonal Antibodies Against Ebola Virus Glycoprotein Generated After Deoxyribonucleic Acid and Protein Vaccination of Transchromosomal Bovines.

J Infect Dis 2018 11;218(suppl_5):S597-S602

Division of Viral Products, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, Maryland.

Several Ebola vaccines and therapeutics are under clinical development. However, limited knowledge exists on the quality of antibody response generated by different Ebola vaccines. In this study, antibody repertoire induced by vaccination of transchromosomal bovine (TcB) with Ebola virus (EBOV) glycoprotein ([GP]; recombinant GP [rGP]) encoded by either deoxyribonucleic acid (DNA) or nanoparticle-based vaccine platform was analyzed using EBOV genome fragment phage display library and surface plasmon resonance (SPR)-based real-time kinetics assay to measure antibody affinity maturation to both native and partially denatured Ebola GP as well as GP containing the receptor binding domain but lacking the mucin-like domain. Immunoglobulin (IgG) obtained from rGP nanoparticle-vaccinated TcB demonstrated ~4-fold higher binding affinity compared with DNA-vaccinated TcB-induced IgG against the native rGP's. The rGP nanoparticle vaccine generated a more robust and diverse antibody immune response to the native EBOV-GP compared with the DNA vaccine, which may explain the protective efficacy observed for these antibody preparations.
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http://dx.doi.org/10.1093/infdis/jiy325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7107430PMC
November 2018

Development and Regulation of Novel Influenza Virus Vaccines: A United States Young Scientist Perspective.

Authors:
Surender Khurana

Vaccines (Basel) 2018 Apr 27;6(2). Epub 2018 Apr 27.

Division of Viral Products, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Silver Spring, MD 20993, USA.

Vaccination against influenza is the most effective approach for reducing influenza morbidity and mortality. However, influenza vaccines are unique among all licensed vaccines as they are updated and administered annually to antigenically match the vaccine strains and currently circulating influenza strains. Vaccine efficacy of each selected influenza virus vaccine varies depending on the antigenic match between circulating strains and vaccine strains, as well as the age and health status of the vaccine recipient. Low vaccine effectiveness of seasonal influenza vaccines in recent years provides an impetus to improve current seasonal influenza vaccines, and for development of next-generation influenza vaccines that can provide broader, long-lasting protection against both matching and antigenically diverse influenza strains. This review discusses a perspective on some of the issues and formidable challenges facing the development and regulation of the next-generation influenza vaccines.
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http://dx.doi.org/10.3390/vaccines6020024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6027304PMC
April 2018

Comparison of the Efficacy of N9 Neuraminidase-Specific Monoclonal Antibodies against Influenza A(H7N9) Virus Infection.

J Virol 2018 02 30;92(4). Epub 2018 Jan 30.

Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA

The fifth wave of A(H7N9) virus infection in China from 2016 to 2017 caused great concern due to the large number of individuals infected, the isolation of drug-resistant viruses, and the emergence of highly pathogenic strains. Antibodies against neuraminidase (NA) provide added benefit to hemagglutinin-specific immunity and may be important contributors to the effectiveness of A(H7N9) vaccines. We generated a panel of mouse monoclonal antibodies (MAbs) to identify antigenic domains on NA of the novel A(H7N9) virus and compared their functional properties. The loop formed in the region of residue 250 (250 loop) and the domain formed by the loops containing residues 370, 400, and 430 were identified as major antigenic regions. MAbs 1E8, 2F6, 10F4, and 11B2, which recognize these two antigenic domains, were characterized in depth. These four MAbs differ in their abilities to inhibit cleavage of small and large substrates (methyl-umbelliferyl-acetyl neuraminic acid [MU-NANA] and fetuin, respectively) in NA inhibition assays. 1E8 and 11B2 did not inhibit NA cleavage of either MU-NANA or fetuin, and 2F6 inhibited cleavage of fetuin alone, whereas 10F4 inhibited cleavage of both substrates. All four MAbs reduced the spread of viruses carrying either the wild-type N9 or N9 with antiviral-resistant mutations but to different degrees. These MAbs have different levels of effectiveness: 10F4 was the most effective in protecting mice against challenge with A(H7N9) virus, 2F6 was less effective, and 11B2 failed to protect BALB/c mice at the doses tested. Our study confirms that NA-specific antibodies can protect against A(H7N9) infection and suggests that properties can be used to rank antibodies with therapeutic potential. The novel A(H7N9) viruses that emerged in China in 2013 continue to infect humans, with a high fatality rate. The most recent outbreak resulted in a larger number of human cases than previous epidemic waves. Due to the absence of a licensed vaccine and the emergence of drug-resistant viruses, there is a need to develop alternative approaches to prevent or treat A(H7N9) infection. We have made a panel of mouse monoclonal antibodies (MAbs) specific for neuraminidase (NA) of A(H7N9) viruses; some of these MAbs are effective in inhibiting viruses that are resistant to antivirals used to treat A(H7N9) patients. Binding avidity, inhibition of NA activity, and plaque formation correlated with the effectiveness of these MAbs to protect mice against lethal A(H7N9) virus challenge. This study identifies measures that can be used to predict the efficacy of NA-specific antibodies, providing a way to select MAbs for further therapeutic development.
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http://dx.doi.org/10.1128/JVI.01588-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5790931PMC
February 2018

A Potent Germline-like Human Monoclonal Antibody Targets a pH-Sensitive Epitope on H7N9 Influenza Hemagglutinin.

Cell Host Microbe 2017 Oct 28;22(4):471-483.e5. Epub 2017 Sep 28.

Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China. Electronic address:

The H7N9 influenza virus causes high-mortality disease in humans but no effective therapeutics are available. Here we report a human monoclonal antibody, m826, that binds to H7 hemagglutinin (HA) and protects against H7N9 infection. m826 binds to H7N9 HA with subnanomolar affinity at acidic pH and 10-fold lower affinity at neutral pH. The high-resolution (1.9 Å) crystal structure of m826 complexed with H7N9 HA indicates that m826 binds an epitope that may be fully exposed upon pH-induced conformational changes in HA. m826 fully protects mice against lethal challenge with H7N9 virus through mechanisms likely involving antibody-dependent cell-mediated cytotoxicity. Interestingly, immunogenetic analysis indicates that m826 is a germline antibody, and m826-like sequences can be identified in H7N9-infected patients, healthy adults, and newborn babies. These m826 properties offer a template for H7N9 vaccine immunogens, a promising candidate therapeutic, and a tool for exploring mechanisms of virus infection inhibition by antibodies.
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http://dx.doi.org/10.1016/j.chom.2017.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6290738PMC
October 2017

What Is the Predictive Value of Animal Models for Vaccine Efficacy in Humans? The Importance of Bridging Studies and Species-Independent Correlates of Protection.

Cold Spring Harb Perspect Biol 2018 04 2;10(4). Epub 2018 Apr 2.

Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993.

Animal models have played a pivotal role in all stages of vaccine development. Their predictive value for vaccine effectiveness depends on the pathogen, the robustness of the animal challenge model, and the correlates of protection (if known). This article will cover key questions regarding bridging animal studies to efficacy trials in humans. Examples include human papillomavirus (HPV) vaccine in which animal protection after vaccination with heterologous prototype virus-like particles (VLPs) predicted successful efficacy trials in humans, and a recent approval of anthrax vaccine in accordance with the "Animal Rule." The establishment of animal models predictive of vaccine effectiveness in humans has been fraught with difficulties with low success rate to date. Challenges facing the use of animal models for vaccine development against Ebola and HIV will be discussed.
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http://dx.doi.org/10.1101/cshperspect.a028902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880166PMC
April 2018

Preclinical evaluation of bacterially produced RSV-G protein vaccine: Strong protection against RSV challenge in cotton rat model.

Sci Rep 2017 02 10;7:42428. Epub 2017 Feb 10.

Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, 20903, USA.

In current study, we evaluated the safety and protective efficacy of recombinant unglycosylated RSV G protein ectodomain produced in E. coli (in presence and absence of oil-in-water adjuvant) in a preclinical RSV susceptible cotton rat challenge model compared to formaldehyde inactivated RSV (FI-RSV) and live RSV experimental infection. The adjuvanted G protein vaccine induced robust neutralization antibody responses comparable to those generated by live RSV infection. Importantly, adjuvanted G protein significantly reduced viral loads in both the lungs and nose at early time points following viral challenge. Antibody kinetics determined by Surface Plasmon Resonance showed that adjuvanted G generated 10-fold higher G-binding antibodies compared to non-adjvuanted G vaccine and live RSV infection, which correlated strongly with both neutralization titers and viral load titers in the nose and lungs post-viral challenge. Antibody diversity analysis revealed immunodominant antigenic sites in the N- and C-termini of the RSV-G protein, that were boosted >10-fold by adjuvant and inversely correlated with viral load titers. Enhanced lung pathology was observed only in animals vaccinated with FI-RSV, but not in animals vaccinated with unadjuvanted or adjuvanted RSV-G vaccine after viral challenge. The bacterially produced unglycosylated G protein could be developed as a protective vaccine against RSV disease.
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http://dx.doi.org/10.1038/srep42428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5301242PMC
February 2017

Development of bioluminescence imaging of respiratory syncytial virus (RSV) in virus-infected live mice and its use for evaluation of therapeutics and vaccines.

Vaccine 2017 01 15;35(4):694-702. Epub 2016 Dec 15.

Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD 20993, USA. Electronic address:

Respiratory Syncytial virus (RSV) is one of the leading causes of pneumonia among infants with no human vaccine or efficient curative treatments. Efforts are underway to develop new RSV vaccines and therapeutics. There is a dire need for animal models for preclinical evaluation and selection of products against RSV. Herein, we developed a whole body bioluminescence imaging to follow replication of RSV A2 virus strain expressing firefly luciferase (RSVA2-line19-FFL) in live BALB/c mice that can be used as an extremely sensitive readout for studying effects of antiviral and vaccines in living mice. Strong bioluminescence signal was detected in the nasal cavity and in the lungs following intranasal infection of mice with RSVA2-line19-FFL. The kinetics of viral replication in lungs quantified by daily live imaging strongly correlated with viral titers measured by ex-vivo plaque assay and by assessing viral RNA by qRT-PCR. Vaccination of mice with a pre-fusion F protein elicited high neutralizing antibody titers conferring strong protective immunity against virus replication in the nasal cavity and lungs. In contrast, post-challenge treatment of mice with the monoclonal antibody Palivizumab two days after infection reduced viral replication in the nasal cavity at day 4, but only modestly reduced virus loads in the lungs by day 5. In contrast to RSV bioluminescence, plaque assay did not detect viral titers in lungs on day 5 in Palivizumab-treated animals. This difference between viral loads measured by the two assays was found to be due to coating of virions with the Palivizumab that blocked infection of target cells in vitro and shows importance of live imaging in evaluation of RSV therapeutics. This recombinant RSV based live imaging animal model is convenient and valuable tool that can be used to study host dissemination of RSV and evaluation of antiviral compounds and vaccines against RSV.
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http://dx.doi.org/10.1016/j.vaccine.2016.11.044DOI Listing
January 2017

Human antibody repertoire after VSV-Ebola vaccination identifies novel targets and virus-neutralizing IgM antibodies.

Nat Med 2016 12 31;22(12):1439-1447. Epub 2016 Oct 31.

Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA.

Development of an effective vaccine against Ebola virus is of high priority. However, knowledge about potential correlates of protection and the durability of immune response after vaccination is limited. Here, we elucidate the human antibody repertoire after administration of vesicular stomatitis virus (VSV)-Ebola vaccine at 3 million, 20 million and 100 million plaque-forming units (PFU) and homologous VSV-Ebola vaccine boost in healthy adult volunteers. Whole genome-fragment phage display libraries, expressing linear and conformational epitopes of Ebola glycoprotein (GP), showed higher diversity of antibody epitopes in individuals vaccinated with 20 million PFU than in those vaccinated with 3 million or 100 million PFU. Surface plasmon resonance kinetics showed higher levels of GP-binding antibodies after a single vaccination with 20 million or 100 million PFU than with 3 million PFU, and these correlated strongly with neutralization titers. A second vaccination did not boost antibody or virus neutralization titers, which declined rapidly, and induced only minimal antibody affinity maturation. Isotype analysis revealed a predominant IgM response even after the second vaccination, which contributed substantially to virus neutralization in vitro. These findings may help identify new vaccine targets and aid development and evaluation of effective countermeasures against Ebola.
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http://dx.doi.org/10.1038/nm.4201DOI Listing
December 2016