A Peptide-Based Assay Discriminates Individual Antibody Response to the COVID-19 Pfizer/BioNTech mRNA Vaccine

The coronavirus disease 2019 (COVID-19) mRNA vaccine developed by Pfizer/BioNTech has been shown to be capable of developing an excellent antibody response against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, with good production of neutralizing antibodies. Herein, we analyzed differences in the antibody response elicited by inoculation of the Pfizer/BioNTech vaccine through a peptide-based enzyme-linked immunosorbent assay (ELISA) that utilizes synthetic peptides derived from the spike protein in the immuno-adsorbent phase. Immunoreactivity against synthetic peptides was measured at different time points from vaccination and was also correlated with the SARS-CoV-2 neutralizing capacity. Our results indicate that all vaccinated subjects except one show reactive antibodies to at least one peptide at both 30 and 60 days after injection of the first dose. Only one of the 19 analyzed subjects showed no antibody response toward any of the selected peptides, consistently with a lower neutralizing capacity. More importantly, our data showed that the antibody response elicited by inoculation of the two doses of the Pfizer vaccine appears to be qualitatively individual, both in the type of recognized peptides and in the temporal persistence of the antibody response. Together with previous published data, our findings suggest that for effective pandemic control, it is important to constantly monitor the antibody protection in the population, and the assay described here could be a valid tool for this purpose.

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Assessment of Mumps Virus-Specific Antibodies: Comparison of Plaque Reduction Neutralization Test and Enzyme-Linked Immunosorbent Assay Estimates

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz345 ◽

2019 ◽

Vol 220 (9) ◽

pp. 1462-1468 ◽

Cited By ~ 3

Author(s):

Stéphanie Ravault ◽

Damien Friel ◽

Emmanuel Di Paolo ◽

Adrian Caplanusi ◽

Paul Gillard ◽

...

Keyword(s):

Antibody Response ◽

Immunosorbent Assay ◽

Neutralizing Antibodies ◽

Neutralization Test ◽

Pearson Correlation ◽

Mumps Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Serological Response ◽

Moderate Correlation ◽

Plaque Reduction Neutralization Test

Abstract Background The plaque reduction neutralization test (PRNT), which measures a subset of immunoglobulin antibodies (functional neutralizing antibodies), and the enzyme-linked immunosorbent assay (ELISA), which measures total immunoglobulin (neutralizing and nonneutralizing antibodies), characterize different aspects of the anti–mumps virus antibody response after vaccination. Methods Data from a recent phase 3 clinical trial (NCT01681992) of 2 measles-mumps-rubella vaccines were used to compare anti-mumps antibody responses measured using an unenhanced PRNT (GSK; seropositivity cutoff and threshold, 2.5 and 4 times the 50% end-point dilution, respectively) with those estimated using an ELISA (thresholds, 5 and 10 ELISA units/mL, respectively). Results Of 3990 initially seronegative samples, 3284 (82.3%) were seropositive after vaccination for anti-mumps antibodies in both assays. The Pearson correlation coefficient for double-positive samples was 0.57, indicative of a moderate correlation. Receiver operating characteristic curve analysis showed that an ELISA threshold of 51.7 ELISA units/mL best corresponded to the PRNT seroresponse threshold. There was no obvious vaccine brand effect on the correlation between assays. Conclusions The moderate correlation between the anti-mumps antibody measurements obtained with PRNT and ELISA reflects different aspects of the serological response. In the absence of a well-defined protective serological threshold, PRNT provides complementary information on the antibody response, whereas ELISA remains a critically useful measurement of vaccine immunogenicity.

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Nasal Immunization of Mice with Human Papillomavirus Type 16 Virus-Like Particles Elicits Neutralizing Antibodies in Mucosal Secretions

Journal of Virology ◽

10.1128/jvi.72.10.8220-8229.1998 ◽

1998 ◽

Vol 72 (10) ◽

pp. 8220-8229 ◽

Cited By ~ 120

Author(s):

Carole Balmelli ◽

Richard Roden ◽

Alexandra Potts ◽

John Schiller ◽

Pierre De Grandi ◽

...

Keyword(s):

Human Papillomavirus ◽

Antibody Response ◽

Neutralizing Antibodies ◽

Secretory Iga ◽

Enzyme Linked Immunosorbent Assay ◽

Virus Like Particles ◽

Human Papillomavirus Type 16 ◽

Specific Igg ◽

Nasal Immunization ◽

Mucosal Secretions

ABSTRACT To specifically induce a mucosal antibody response to purified human papillomavirus type 16 (HPV16) virus-like particles (VLP), we immunized female BALB/c mice orally, intranasally, and/or parenterally and evaluated cholera toxin (CT) as a mucosal adjuvant. Anti-HPV16 VLP immunoglobulin G (IgG) and IgA titers in serum, saliva, and genital secretions were measured by enzyme-linked immunosorbent assay (ELISA). Systemic immunizations alone induced HPV16 VLP-specific IgG in serum and, to a lesser extent, in genital secretions but no secretory IgA. Oral immunization, even in the presence of CT, was inefficient. However, three nasal immunizations with 5 μg of VLP given at weekly intervals to anesthetized mice induced high (>104) and long-lasting (>15 weeks) titers of anti-HPV16 VLP antibodies in all samples, including IgA and IgG in saliva and genital secretions. CT enhanced the VLP-specific antibody response 10-fold in serum and to a lesser extent in saliva and genital secretions. Nasal immunization of conscious mice compared to anesthetized mice was inefficient and correlated with the absence of uptake of a marker into the lung. However, a 1-μg VLP systemic priming followed by two 5-μg VLP intranasal boosts in conscious mice induced both HPV16 VLP-specific IgG and IgA in secretions, although the titers were lower than in anesthetized mice given three intranasal immunizations. Antibodies in serum, saliva, and genital secretions of immunized mice were strongly neutralizing in vitro (50% neutralization with ELISA titers of 65 to 125). The mucosal and systemic/mucosal HPV16 VLP immunization protocols that induced significant titers of neutralizing IgG and secretory IgA in mucosal secretions in mice may be relevant to genital HPV VLP-based human vaccine trials.

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Potent anti-SARS-CoV-2 Antibody Responses are Associated with Better Prognosis in Hospital Inpatient COVID-19 Disease

10.1101/2020.08.22.20176834 ◽

2020 ◽

Author(s):

Patrick J Tighe ◽

Richard A Urbanowicz ◽

Lucy Fairclough ◽

C Patrick McClure ◽

Brian J Thomson ◽

...

Keyword(s):

Antibody Response ◽

Neutralizing Antibodies ◽

Clinical Intervention ◽

Neutralizing Antibody ◽

Antibody Responses ◽

Spike Protein ◽

Effective Vaccine ◽

Hospital Inpatient ◽

Protein Variant ◽

Immune Correlates

COVID-19 continues to cause a pandemic, having infected more than 20 million people globally. Successful elimination of the SARS-CoV-2 virus will require an effective vaccine. However, the immune correlates of infection are currently poorly understood. While neutralizing antibodies are believed to be essential for protection against infection, the contribution of the neutralizing antibody response to resolution of SARS-CoV-2 infection has not yet been defined. In this study the antibody responses to the SARS-CoV-2 spike protein and nucleocapsid proteins were investigated in a UK patient cohort, using optimised immunoassays and a retrovirus-based pseudotype entry assay. It was discovered that in severe COVID-19 infections an early antibody response to both antigens was associated with improved prognosis of infection. While not all SARS-CoV-2-reactive sera were found to possess neutralizing antibodies, neutralizing potency of sera was found to be greater in patients who went on to resolve infection, compared with those that died from COVID-19. Furthermore, viral genetic variation in spike protein was found to influence the production of neutralizing antibodies. Infection with the recently described spike protein variant 614G produced higher levels of neutralizing antibodies when compared to viruses possessing the 614D variant. These findings support the assertion that vaccines targeting generation of neutralizing antibodies may be useful at limiting SARS-CoV-2 infection. Assessment of the antibody responses to SARS-CoV-2 at time of diagnosis will be a useful addition to the diagnostic toolkit, enabling stratification of clinical intervention for severe COVID-19 disease.

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

Science Translational Medicine ◽

10.1126/scitranslmed.abc3539 ◽

2020 ◽

Vol 12 (550) ◽

pp. eabc3539 ◽

Cited By ~ 27

Author(s):

Supriya Ravichandran ◽

Elizabeth M. Coyle ◽

Laura Klenow ◽

Juanjie Tang ◽

Gabrielle Grubbs ◽

...

Keyword(s):

Amino Acids ◽

Neutralizing Antibodies ◽

Antibody Responses ◽

Spike Protein ◽

Enzyme Linked Immunosorbent Assay ◽

Limited Information ◽

Protein Antigens ◽

Phage Display Libraries ◽

Rational Vaccine Design ◽

And Function

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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Generation of Neutralizing Antipeptide Antibodies to the Enzymatic Domain of Pseudomonas aeruginosa Exotoxin A

Infection and Immunity ◽

10.1128/iai.66.5.2170-2179.1998 ◽

1998 ◽

Vol 66 (5) ◽

pp. 2170-2179 ◽

Cited By ~ 10

Author(s):

Haissam S. Elzaim ◽

Ashok K. Chopra ◽

Johnny W. Peterson ◽

Rick Goodheart ◽

John P. Heggers

Keyword(s):

Pseudomonas Aeruginosa ◽

Topical Treatment ◽

Synthetic Peptides ◽

Neutralizing Antibodies ◽

Opportunistic Infections ◽

Therapeutic Potential ◽

Passive Immunization ◽

Enzyme Linked Immunosorbent Assay ◽

Burn Patients ◽

Specific Antibodies

ABSTRACT Burn patients suffer a break in the physical barrier (skin), which, when combined with their generalized state of immunodeficiency, creates an open window for opportunistic infections, mainly withPseudomonas aeruginosa. Infection of the burn wound has always been a major factor in retardation of wound healing, and sepsis remains the leading cause of death in burn patients. Because studies have shown that topical treatment with antiexotoxin A (ETA) antibodies significantly increases survival in rats infected with toxin-producing strains of P. aeruginosa, we examined 11 synthetic peptides encompassing 12 to 45 amino acid (aa) residues, representing what were predicted by computer analysis to be the most hydrophilic and antigenic regions of ETA. These synthetic peptides were injected into rabbits for antibody production. Different groups of rabbits were immunized with a combination of peptides, with each combination representing one of the three distinct domains of ETA. Animals immunized with various peptide combinations produced peptide-specific antibodies that exhibited cross-reactivity to ETA. Two major epitopes were identified on the ETA molecule by experiments with peptide-specific antibodies in enzyme-linked immunosorbent assay and immunoprecipitation. One of these epitopes was located in the translocation domain (II) (aa 297 to 310), while the other was mapped to the last 13 aa residues at the carboxy-terminal end of the enzymatic domain (III) (aa 626 to 638). Of these two regions, the epitope in the enzymatic domain induced a much higher level of neutralizing antibodies that abrogated the cytotoxic activity of ETA in vitro. Antibodies to this epitope blocked the ADP-ribosyltransferase activity of ETA and appeared to interfere with binding of the substrate elongation factor 2 to the enzymatic active site of the ETA molecule. We conclude that polyclonal, as well as monoclonal, antibodies to short peptides, representing small regions of ETA, may have therapeutic potential in passive immunization or topical treatment of burn patients infected with toxin-producing strains of P. aeruginosa.

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Robust neutralizing antibodies to SARS-CoV-2 infection persist for months

Science ◽

10.1126/science.abd7728 ◽

2020 ◽

Vol 370 (6521) ◽

pp. 1227-1230 ◽

Cited By ~ 59

Author(s):

Ania Wajnberg ◽

Fatima Amanat ◽

Adolfo Firpo ◽

Deena R. Altman ◽

Mark J. Bailey ◽

...

Keyword(s):

New York ◽

New York City ◽

Severe Acute Respiratory Syndrome ◽

Antibody Response ◽

York City ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Antibody Responses ◽

Spike Protein ◽

Global Pandemic

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic with millions infected and more than 1 million fatalities. Questions regarding the robustness, functionality, and longevity of the antibody response to the virus remain unanswered. Here, on the basis of a dataset of 30,082 individuals screened at Mount Sinai Health System in New York City, we report that the vast majority of infected individuals with mild-to-moderate COVID-19 experience robust immunoglobulin G antibody responses against the viral spike protein. We also show that titers are relatively stable for at least a period of about 5 months and that anti-spike binding titers significantly correlate with neutralization of authentic SARS-CoV-2. Our data suggest that more than 90% of seroconverters make detectable neutralizing antibody responses. These titers remain relatively stable for several months after infection.

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Successful Anti-SARS-CoV-2 Spike Protein Antibody Response to Vaccination in MAGT1 Deficiency

Allergy & Rhinology ◽

10.1177/21526567211056239 ◽

2021 ◽

Vol 12 ◽

pp. 215265672110562

Author(s):

Maaz Jalil ◽

Marija Rowane ◽

Jayanth Rajan ◽

Robert Hostoffer

Keyword(s):

Antibody Response ◽

Messenger Rna ◽

Neutralizing Antibodies ◽

Office Visit ◽

Spike Protein ◽

Antibody Detection ◽

Epstein Barr Virus Infection ◽

Protein Antigens ◽

Antibody Testing ◽

Post Vaccination

Background Novel messenger RNA vaccines against severe acute respiratory syndrome coronavirus (SARS-CoV-2) have been vital in resolving the coronavirus disease-2019 (COVID-19) pandemic. Detection of neutralizing antibodies (NAbs) against the SARS-CoV-2 spike protein (S) confirms immunogenicity with high sensitivity and specificity. Few recent studies with primary and secondary immunodeficient cohorts present adequate or reduced antibody response. We describe the first reported successful response to anti-SARS-CoV-2 S antibody post-vaccination in magnesium transporter 1 (MAGT1) gene deficiency, more commonly recognized as x-linked immunodeficiency with magnesium defect, Epstein-Barr Virus infection, and neoplasia (XMEN). Case Presentation We present a 30-year-old male with selective anti-polysaccharide antibody deficiency, peripheral blood CD5 + /CD19 + B-cell predominance (97%), MAGT1 mutation, and reduced CD16 + CD56 + natural killer- and/or CD8 + T-cell receptor, Group 2, Member D expression. His initial immunological evaluation revealed all seronegative post-vaccination antibody titers but clinically adequate response to protein antigens tetanus and diphtheria anti-toxoids. COVID-19 vaccination and associated serology antibody testing was recommended at this office visit. Anti-SARS-CoV-2 immunoglobulin (Ig)M and IgG antibodies before and after the first BNT162b2 mRNA COVID-19 vaccine doses, as well as nucleocapsid antibody, were negative. S protein total antibody was reactive after the second dose. Discussion Robust immunological sequelae post-COVID-19 vaccination in the general population are well-documented in the recent literature. Few studies have evaluated COVID-19 vaccination antibody response in immunodeficient patients. The majority positive anti-S antibody detection in most primary immunodeficient (PID) patients among the few studies in the literature, such as the present case, support the safety and efficacy of mRNA COVID-19 vaccination in immunodeficient patients, although larger scale studies are needed. Conclusion We demonstrate successful vaccination in the PID MAGT1 deficiency in this first reported case of reactive anti-S antibody post-COVID-19 vaccination.

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Frequency and Domain Specificity of Toxin-Neutralizing Paratopes in the Human Antibody Response to Anthrax Vaccine Adsorbed

Infection and Immunity ◽

10.1128/iai.01254-08 ◽

2009 ◽

Vol 77 (5) ◽

pp. 2030-2035 ◽

Cited By ~ 24

Author(s):

Donald Reason ◽

Justine Liberato ◽

Jinying Sun ◽

Wendy Keitel ◽

Jianhui Zhou

Keyword(s):

Antibody Response ◽

Neutralizing Antibodies ◽

Protective Antigen ◽

Passive Immunization ◽

Enzyme Linked Immunosorbent Assay ◽

Human Antibody ◽

Anthrax Vaccine ◽

Amino Terminal ◽

Efficacious Vaccine

ABSTRACT Protective antigen (PA) is the cell surface recognition unit of the binary anthrax toxin system and the primary immunogenic component in both the current and proposed “next-generation” anthrax vaccines. Several studies utilizing animal models have indicated that PA-specific antibodies, acquired by either active or passive immunization, are sufficient to protect against infection with Bacillus anthracis. To investigate the human antibody response to anthrax immunization, we have established a large panel of human PA-specific monoclonal antibodies derived from multiple individuals vaccinated with the currently approved anthrax vaccine BioThrax. We have determined that although these antibodies bind PA in standard binding assays such as enzyme-linked immunosorbent assay, Western blotting, capture assays, and dot blots, less than 25% are capable of neutralizing lethal toxin (LT) in vitro. Nonneutralizing antibodies also fail to neutralize toxin when present in combination with other nonneutralizing paratopes. Although neutralizing antibodies recognize determinants throughout the PA monomer, they are significantly less common among those paratopes that bind to the immunodominant amino-terminal portion of the molecule. These findings demonstrate that PA binding alone is not sufficient to neutralize LT and suggest that for an antibody to effectively block PA-mediated toxicity, it must bind to PA such that one of the requisite toxin functions is disrupted. A vaccine design strategy that directed a higher percentage of the antibody response toward neutralizing epitopes may result in a more efficacious vaccine for the prevention of anthrax infection.

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Epitope Mapping of Human Anti-Adeno-Associated Virus Type 2 Neutralizing Antibodies: Implications for Gene Therapy and Virus Structure

Journal of Virology ◽

10.1128/jvi.74.4.1761-1766.2000 ◽

2000 ◽

Vol 74 (4) ◽

pp. 1761-1766 ◽

Cited By ~ 164

Author(s):

Marina Moskalenko ◽

Lili Chen ◽

Melinda van Roey ◽

Brian A. Donahue ◽

Richard O. Snyder ◽

...

Keyword(s):

Gene Therapy ◽

Antibody Response ◽

Virus Type ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Factor Ix ◽

Enzyme Linked Immunosorbent Assay ◽

Adeno Associated Virus ◽

Human Factor Ix

ABSTRACT Recombinant adeno-associated virus type 2 (AAV) is a common vector used in human gene therapy protocols. We characterized the humoral immune response to AAV and observed that 80% of normal human subjects have anti-AAV antibodies and that 18% have neutralizing antibodies. To analyze the effect of neutralizing antibodies on AAV readministration, we attempted to deliver recombinant AAV expressing human factor IX (AAV-hFIX) intraportally into the livers of mice which had been preexposed to AAV and shown to harbor a neutralizing antibody response. While all naive control mice expressed hFIX following administration of AAV-hFIX, none of the mice with preexisting immunity expressed hFIX, even after transient immunosuppression at the time of the second administration with anti-CD4 or anti-CD40L antibodies. This suggests that preexisting immunity to AAV, as measured by a neutralizing antibody response, may limit AAV-mediated gene delivery. Using human sera in an enzyme-linked immunosorbent assay for AAV and a capsid peptide scan library to block antibody binding, we mapped seven regions of the AAV capsid containing immunogenic epitopes. Using pools of these peptides to inhibit the binding of neutralizing antibodies, we have identified a subset of six peptides which potentially reconstitute a single neutralizing epitope. This information may allow the design of reverse genetic approaches to circumvent the preexisting immunity that can be encountered in some individuals.

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Antibody repertoire induced by SARS-CoV-2 spike protein immunogens

10.1101/2020.05.12.091918 ◽

2020 ◽

Cited By ~ 8

Author(s):

Supriya Ravichandran ◽

Elizabeth M. Coyle ◽

Laura Klenow ◽

Juanjie Tang ◽

Gabrielle Grubbs ◽

...

Keyword(s):

Antibody Response ◽

Neutralizing Antibodies ◽

Neutralization Assay ◽

Spike Protein ◽

Antibody Repertoire ◽

Limited Information ◽

Protein Antigens ◽

Phage Display Libraries ◽

Rational Vaccine Design ◽

And Function

ABSTRACTMultiple vaccine candidates against SARS-CoV-2 based on viral spike protein are under development. However, there is limited information on the quality of antibody response generated following vaccination by these vaccine modalities. To better understand antibody response induced by spike protein-based vaccines, we immunized rabbits with various SARS-CoV-2 spike protein antigens: S-ectodomain (S1+S2) (aa 16-1213), which lacks the cytoplasmic and transmembrane domains (CT-TM), the S1 domain (aa 16-685), the receptor-binding domain (RBD) (aa 319-541), and the S2 domain (aa 686-1213 as control). Antibody response was analyzed by ELISA, Surface Plasmon Resonance (SPR) against different Spike proteins in native conformation, and a pseudovirion neutralization assay to measure the quality and function of the antibodies elicited by the different Spike antigens. All three antigens (S1+S2 ectodomain, S1 domain, and RBD) 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 surprisingly the RBD immunogen elicited a higher antibody titer with 5-fold higher affinity antibodies to native spike antigens compared with other spike antigens. These findings may help guide rational vaccine design and facilitate development and evaluation of effective therapeutics and vaccines against COVID-19 disease.One Sentence SummarySARS-CoV-2 Spike induced immune response

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